Human cyclic nucleotide PDES

ABSTRACT

The invention provides human cyclic nucleotide phosphodiesterases (HSPDE10A) and polynucleotides which identify and encode HSPDE10A. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists and methods for diagnosing or treating disorders associated with expression of HSPDE10A.

This application is a continuation-in-part of U.S. Ser. No. 09/226,741filed Jan. 7, 1999, now U.S. Pat. No. 6,100,037.

FIELD OF THE INVENTION

This invention relates to nucleic acid and amino acid sequences of humancyclic nucleotide phosphodiesterases and their mammalian variants and tothe use of these sequences in the diagnosis and treatment of cancer andimmune disorders.

BACKGROUND OF THE INVENTION

Cyclic nucleotides (cAMP and cGMP) function as intracellular secondmessengers to transduce a variety of extracellular signals includinghormones, light, and neurotransmitters. Cyclic nucleotidephosphodiesterases (PDEs) degrade cyclic nucleotides to thecorresponding monophosphates, thereby regulating the intracellularconcentrations of cyclic nucleotides and their effects on signaltransduction. At least seven families of mammalian PDEs have beenidentified based on substrate specificity and affinity, sensitivity tocofactors, and sensitivity to inhibitory drugs (Beavo (1995) Physiol Rev75:725-748). Several of these families contain distinct genes, many ofwhich are expressed in different tissues as splice variants. Withinfamilies, there are multiple isozymes and multiple splice variants ofthose isozymes. The existence of multiple PDE families, isozymes, andsplice variants presents an opportunity for regulation of cyclicnucleotide levels and functions.

Type 1 PDEs (PDE1s) are Ca²⁺/calmodulin-dependent and appear to beencoded by three different genes, each having at least two differentsplice variants. PDE1s have been found in the lung, heart, and brain.Some of the Ca²⁺/calmodulin-dependent PDEs are regulated in vitro byphosphorylation and dephosphorylation. Phosphorylation of PDE1 decreasesthe affinity of the enzyme for calmodulin, decreases PDE activity, andincreases steady state levels of cAMP. PDE2s are cGMP stimulated PDEsthat are localized in the brain and are thought to mediate the effectsof cAMP on catecholamine secretion. PDE3s are one of the major familiesof PDEs present in vascular smooth muscle. PDE3s are inhibited by cGMP,have high specificity for cAMP as a substrate, and play a role incardiac function. One isozyme of PDE3 is regulated by one or moreinsulin-dependent kinases. PDE4s are the predominant isoenzymes in mostinflammatory cells, and some PDE4s are activated by cAMP-dependentphosphorylation. PDE5s are thought to be cGMP specific but may alsohydrolyze cAMP. High levels of PDE5s are found in most smooth musclepreparations, in platelets, and in the kidney. PDE6s play a role invision and are regulated by light and cGMP. The PDE7 class, consistingof only one known member, is cAMP-specific and is most closely relatedto PDE4. PDE7 is not inhibited by rolipram, a specific inhibitor of PDE4(Beavo, supra). PDE8 and PDE9 represent two new families of PDEs. PDE8sare cAMP specific, most closely related to PDE4, insensitive torolipram, and sensitive to dipyridimole. PDE9s are cGMP specific andsensitive only to the PDE inhibitor, zaprinast.

PDEs are composed of a catalytic domain of about 270 amino acids, anN-terminal regulatory domain responsible for binding cofactors, and, insome cases, a C-terminal domain of unknown function. A conserved motif,HDXXHXGXXN, has been identified in the catalytic domain of all PDEs. InPDE5, an N-terminal cGMP binding domain spans about 380 amino acidresidues and comprises tandem repeats of the conserved sequence motifN(R/K)XnFX₃DE (McAllister-Lucas et al. (1993) J Biol Chem268:22863-22873). The NKXnD motif has been shown by mutagenesis to beimportant for cGMP binding (Turko et al. (1996) J Biol Chem271:22240-22244). PDE families display approximately 30% amino acididentity within the catalytic domain; however, isozymes within the samefamily typically display about 85-95% identity in this region (e.g.PDE4A vs PDE4B). Furthermore, within a family there is extensivesequence similarity (>60%) outside the catalytic domain; while acrossfamilies, there is little or no sequence similarity.

Many functions of immune and inflammatory responses are inhibited byagents that increase intracellular levels of cAMP (Verghese et al.(1995) Mol Pharmacol 47:1164-1171). A variety of diseases have beenattributed to increased PDE activity and associated with decreasedlevels of cyclic nucleotides. A form of diabetes insipidus in the mousehas been associated with increased PDE4 activity, and an increase inlow-K_(m) cAMP PDE activity has been reported in leukocytes of atopicpatients. Defects in PDEs have also been associated with retinaldisease. Retinal degeneration in the rd mouse, autosomal recessiveretinitis pigmentosa in humans, and rod/cone dysplasia 1 in Irish Setterdogs have been attributed to mutations in the PDE6B gene. PDE3 has beenassociated with cardiac disease.

Many inhibitors of PDEs have been identified and have undergone clinicalevaluation. PDE3 inhibitors are being developed as antithromboticagents, antihypertensive agents, and as cardiotonic agents useful in thetreatment of congestive heart failure. Rolipram, a PDE4 inhibitor, hasbeen used in the treatment of depression, and other inhibitors of PDE4are undergoing evaluation as anti-inflammatory agents. Rolipram has alsobeen shown to inhibit lipopolysaccharide induced TNF-α which has beenshown to enhance HIV-1 replication in vitro. Therefore, rolipram mayinhibit HIV-1 replication (Angel et al. (1995) AIDS 9:1137-44).Additionally, rolipram, based on its ability to suppress the productionof cytokines ,such as TNF-α and β and interferon γ, has been shown to beeffective in the treatment of encephalomyelitis. Rolipram may also beeffective in treating tardive dyskinesia and was effective in treatingmultiple sclerosis in an experimental animal model (Sommer et al. (1995)Nature Med 1:244-248; Sasaki et al. (1995) Eur J Pharmacol 282:71-76).

Theophylline is a nonspecific PDE inhibitor used in the treatment ofbronchial asthma and other respiratory diseases. Theophylline isbelieved to act on airway smooth muscle function and in ananti-inflamatory or immunomodulatory capacity in the treatment ofrespiratory diseases (Banner and Page (1995) Eur Respir J 8:996-1000).Pentoxifylline is another nonspecific PDE inhibitor used in thetreatment of intermittent claudication and diabetes-induced peripheralvascular disease. Pentoxifylline is also known to block TNF-α productionand may inhibit HIV-1 replication (Angel, supra).

PDEs have also been reported to effect cellular proliferation of avariety of cell types and have been implicated in various cancers. Banget al. (1994; Proc Natl Acad Sci 91:5330-5334) reported that growth ofprostate carcinoma cell lines DU 145 and LNCaP was inhibited by deliveryof cAMP derivatives and phosphodiesterase inhibitors. These cells alsoshowed a permanent conversion in phenotype from epithelial to neuronalmorphology. Others have suggested that PDE inhibitors have the potentialto regulate mesangial cell proliferation and lymphocyte proliferation(Matousovic et al. (1995) J Clin Invest 96:401-410; Joulain et al.(1995) J Lipid Mediat Cell Signal 11:63-79, respectively). Finally,Deonarain et al.(1994; Br J Cancer 70:786-94) describe a cancertreatment that involves intracellular delivery of phosphodiesterases toparticular cellular compartments of tumors which results in cell death.

The discovery of a new human cyclic nucleotide phosphodiesterase and thepolynucleotides encoding them satisfies a need in the art by providingnew compositions which are useful in the diagnosis and treatment ofcancer and immune disorders.

SUMMARY OF THE INVENTION

The invention is based on the discovery of new human cyclic nucleotidephosphodiesterases referred to collectively as “HSPDE10A” andindividually as “HSPDE10A ” and “HSPDE10A2”, the polynucleotidesencoding HSPDE10A1 and HSPDE10A2, and the use of these compositions forthe diagnosis and treatment of cancer and immune disorders.

The invention features a purified protein comprising the amino acidsequence of SEQ ID NO:1, SEQ ID NO:3, or portions thereof. The inventionprovides a purified variant having at least 90% amino acid sequenceidentity to the amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, orportions thereof.

The invention provides an isolated polynucleotide encoding the proteincomprising the amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, orportions thereof. The invention also provides an isolated polynucleotidewhich hybridizes under stringent conditions to the polynucleotideencoding the protein comprising the amino acid sequence of SEQ ID NO:1,SEQ ID NO:3, or portions thereof. The invention further provides anisolated polynucleotide having a sequence which is complementary to thepolynucleotide encoding the protein comprising the amino acid sequenceof SEQ ID NO:1, SEQ ID NO:3, or portions thereof.

The invention provides an isolated polynucleotide comprising the nucleicacid sequence of SEQ ID NO:2, SEQ ID NO:4, or fragments thereof andisolated variants having at least 70% polynucleotide sequence identityto the polynucleotide comprising the polynucleotide sequence of SEQ IDNO:2, SEQ ID NO:4, or fragments thereof. The invention also provides anisolated polynucleotide having a sequence complementary to thepolynucleotide comprising the polynucleotide sequence of SEQ ID NO:2,SEQ ID NO:4, or fragments thereof.

The invention provides a method for detecting a polynucleotide in asample containing nucleic acids comprising hybridizing the complement ofthe polynucleotide to at least one of the nucleic acid of the sample,thereby forming a hybridization complex, and detecting the hybridizationcomplex, wherein the presence of the hybridization complex indicates thepresence of the polynucleotide in the sample. In one aspect, the methodfurther comprises amplifying the nucleic acids of the sample prior tohybridization.

The invention also provides an expression vector containing thepolynucleotide encoding the protein comprising the amino acid sequenceof SEQ ID NO:1 or SEQ ID NO:3. In one aspect, the expression vector iscontained within a host cell. The invention further provides a methodfor using a polynucleotide to produce a protein comprising culturing thehost cell under conditions for expression of the protein and recoveringthe protein from the culture. The invention further provides acomposition comprising a purified protein having the amino acid sequenceof SEQ ID NO:1, SEQ ID NO:3, or portions thereof in conjunction with apharmaceutical carrier. The invention also provides a method of usingthe protein to screen a plurality of molecules or compounds to identifya ligand comprising combining the protein with the molecules orcompounds under conditions to allow specific binding and detectingspecific binding, thereby identifying a ligand which specifically bindsthe protein. In one aspect, the molecules and compounds are selectedfrom the group consisting of DNA molecules, RNA molecules, peptidenucleic acid molecules, peptides, proteins, agonists, antagonists,inhibitors, drug compounds and pharmaceutical agents. The inventionfurther provides a composition comprising a purified agonist whichspecifically binds the protein having the amino acid sequence of SEQ IDNO:1, SEQ ID NO:3, or portions thereof.

The invention provides methods for using the protein to producepolyclonal or monoclonal antibodies which specifically bind the protein.The invention also provides a purified antibody which specifically bindsto a protein comprising the amino acid sequence of SEQ ID NO:1 or SEQ IDNO:3. The invention further provides a method for using the antibody inan assay for adenofibromatous hyperplasia of the prostate comprisingcontacting the antibody with a sample from a patient, detecting complexformation between the antibody and protein, comparing complex formationwith a standard, wherein the difference in complex formation indicatesthe presence of the disease.

The invention provides a method for treating adenofibromatoushyperplasia of the prostate as it is associated with decreasedexpression or activity of HSPDE10A comprising administering to a subjectin need of such treatment a composition comprising a purified proteinhaving the amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, or portionsthereof, in conjunction with a pharmaceutical carrier. The inventionalso provides a method for treating adenofibromatous hyperplasia of theprostate comprising administering to a subject in need of such treatmenta composition comprising an agonist which specifically binds the proteinhaving the amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, or portionsthereof

The invention also provides a method for treating a disorder associatedwith increased expression or activity of HSPDE10A, the method comprisingadministering to a subject in need of such treatment an antagonist ofthe protein having the amino acid sequence of SEQ ID NO:1, SEQ ID NO:3,or portions thereof.

BRIEF DESCRIPTION OF THE FIGURES AND TABLE

FIGS. 1A, 1B, 1C, 1D, and 1E show the amino acid sequence (SEQ ID NO:1)and nucleic acid sequence (SEQ ID NO:2) of HSPDE10A1. The alignment wasproduced using MACDNASIS PRO software (Hitachi Software Engineering,South San Francisco, Calif.).

FIGS. 2A, 2B, 2C, 2D, 2E, and 2F show the amino acid sequence (SEQ IDNO:3) and nucleic acid sequence (SEQ ID NO:4) of HSPDE10A2. Thealignment was produced using MACDNASIS PRO software.

FIGS. 3A, 3B, 3C, 3D, and 3E show the amino acid sequence alignmentsbetween HSPDE10A1 (SEQ ID NO:1), HSPDE10A2 (SEQ ID NO:3), and humanPDE5, HPDE5A1 (g3355606; SEQ ID NO:5), produced using the MEGALIGNprogram (DNASTAR, Madison, Wis.).

FIGS. 4A and 4B show the activity assay for HSPDE10A1 using cAMP andcGMP as substrates, respectively. The positive X axis represents thesubstrate concentration (mM), and the positive Y axis represents thereaction velocity in pmoles/minute/ml enzyme. K_(m) and V_(max) valuesfor the enzyme activity with each substrate were calculated from aMichaelis-Menten plot using the “Fit Curve” Microsoft Excel extensionprogram.

FIGS. 5A and 5B show the membrane-based northern analysis of HSPDE10Aexpression in human tissues. The X axis presents the various tissuesanalyzed, and the Y axis presents various size markers. The arrowindicates the location of the major (˜7.5 kb) transcript of HSPDE10A.

FIG. 6 shows the expression of full length HSPDE10A1 in Sf9 cells(arrow; predicted molecular weight ˜56 kDa). Lane 1 shows various sizemarkers and their molecular weights. Lanes 2 and 4, infected cells at64,000 and 12,800 cell equivalents, respectively, show HSPDE10A1. Lanes3 and 5, mock-infected cells at 64,000 and 12,800 cell equivalents,respectively, fail to show the presence of HSPDE10A1.

FIG. 7 shows the northern analysis for HSPDE10A1 produced using theLIFESEQ Gold database (Incyte Genomics, Palo Alto, Calif.). In the firstrange, the first column presents the tissue categories; the secondcolumn, the number of clones in the tissue category; the third column,the number of libraries in which at least one transcript was found; thefourth column, absolute abundance of the transcript; and the fifthcolumn, percent abundance of the trancript. In the second range, thefirst column lists the library name, the second column, the number ofclones sequenced for that library; the third column, description of thetissue; the fourth column, absolute abundance of the transcript; and thefifth column, percent abundance of the trancript.

Table 1 shows the effects of various PDE inhibitors on the activity ofHSPDE10A1. Assays were carried out using cGMP as a substrate at aconcentration of 0.17 mM, equal to ˜⅓ of the K_(m) of cGMP. Inhibitorswere tested over a range of concentrations from ˜0.5 to ˜110 mM. IC₅₀(or K_(i)) values were extrapolated from the dose response curves.

Table 2 shows the programs, their descriptions, references, andthreshold parameters used to analyze HSPDE10A.

DESCRIPTION OF THE INVENTION

Before the present proteins, polynucleotides, and methods are described,it is understood that this invention is not limited to the particularmachines, materials and methods described as these may vary. It is alsoto be understood that the terminology used herein is for the purpose ofdescribing particular embodiments only and is not intended to limit thescope of the present invention which will be limited only by theappended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. For example, a reference to “a hostcell” includes a plurality of such host cells, and a reference to “anantibody” is a reference to one or more antibodies and equivalentsthereof known to those of skill in the art.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any machines,materials, and methods similar or equivalent to those described hereincan be used to practice or test the present invention, the preferredmachines, materials and methods are now described. All patents andpublications mentioned herein are hereby incorporated by reference andhave been cited for the purpose of describing and disclosing the celllines, protocols, reagents and vectors which are reported in thepublications and which might be used in connection with the invention.Nothing herein is to be construed as an admission that the invention isnot entitled to antedate such disclosure by virtue of prior invention.

DEFINITIONS

“HSPDE10A” refers to the amino acid sequences of a purified HSPDE10Aobtained from any species including bovine, ovine, porcine, murine,equine, rodent, and preferably the human species, from any source,whether natural, synthetic, semi-synthetic, or recombinant.

“Array” refers to an ordered arrangement of at least two polynucleotideson a substrate. The arrangement of from about two to about 40,000polynucleotides on the substrate assures that the size and signalintensity of each labeled hybridization complex formed between apolynucleotide and a sample nucleic acid is individuallydistinguishable.

The “complement” of a polynucleotide of the Sequence Listing refers to anucleic acid molecule which is completely complementary over its fulllength and which will hybridize to the polynucleotide or an mRNA underconditions of high stringency.

“Polynucleotide” refers to an isolated cDNA, nucleic acid molecule, orany fragment or complement thereof. It may have originated recombinantlyor synthetically, be double-stranded or single-stranded, representcoding and/or noncoding sequence, an exon with or without an intron froma genomic DNA molecule.

The phrase “polynucleotide encoding a protein” refers to a nucleic acidsequence that closely aligns with sequences which encode conservedregions, motifs or domains that were identified by employing analyseswell known in the art. These analyses include BLAST (Basic LocalAlignment Search Tool; Altschul (1993) J Mol Evol 36: 290-300; Altschulet al. (1990) J Mol Biol 215:403-410) which provides identity within theconserved region. Brenner et al. (1998; Proc Natl Acad Sci 95:6073-6078)who analyzed BLAST for its ability to identify structural homologs bysequence identity found 30% identity is a reliable threshold forsequence alignments of at least 150 residues and 40% is a reasonablethreshold for alignments of at least 70 residues (Brenner et al., p.6076, column 2).

“Derivative” refers to a polynucleotide or a protein that has beensubjected to a chemical modification. Derivatization of a polynucleotidecan involve substitution of a nontraditional base such as queosine or ofan analog such as hypoxanthine. These substitutions are well known inthe art. Derivatization of a protein involves the replacement of ahydrogen by an acetyl, acyl, alkyl, amino, formyl, or morpholino group.Derivative molecules retain the biological activities of the naturallyoccurring molecules but may confer advantages such as longer lifespan orenhanced activity.

“Differential expression” refers to an increased, upregulated orpresent, or decreased, downregulated or absent, gene expression asdetected by the absence, presence, or at least two-fold changes in theamount of transcribed messenger RNA or translated protein in a sample.

“Disorder” refers to conditions, diseases or syndromes in which thepolynucleotides and HSPDE10A are differentially expressed such asadenofibromatous hyperplasia of the prostate.

“Fragment” refers to a chain of consecutive nucleotides from about 61 toabout 5000 base pairs in length. Fragments may be used in PCR orhybridization technologies to identify related nucleic acid moleculesand in binding assays to screen for a ligand. Nucleic acids and theirligands identified in this manner are useful as therapeutics to regulatereplication, transcription or translation.

A “hybridization complex” is formed between a polynucleotide and anucleic acid of a sample when the purines of one molecule hydrogen bondwith the pyrimidines of the complementary molecule, e.g., 5′-A-G-T-C-3′base pairs with 3′-T-C-A-G-5′. The degree of complementarity and the useof nucleotide analogs affect the efficiency and stringency ofhybridization reactions.

“Ligand” refers to any agent, molecule, or compound which will bindspecifically to a complementary site on a cDNA molecule orpolynucleotide, or to an epitope or a protein. Such ligands stabilize ormodulate the activity of polynucleotides or proteins and may be composedof inorganic or organic substances including nucleic acids, proteins,carbohydrates, fats, and lipids.

“Oligonucleotide” refers a single stranded molecule from about 18 toabout 60 nucleotides in length which may be used in hybridization oramplification technologies or in regulation of replication,transcription or translation. Substantially equivalent terms areamplimer, primer, and oligomer.

“Portion” refers to any part of a protein used for any purpose; butespecially, to an epitope for the screening of ligands or for theproduction of antibodies.

“Post-translational modification” of a protein can involve lipidation,glycosylation, phosphorylation, acetylation, racemization, proteolyticcleavage, and the like. These processes may occur synthetically orbiochemically. Biochemical modifications will vary by cellular location,cell type, pH, enzymatic milieu, and the like.

“Probe” refers to a polynucleotide that hybridizes to at least onenucleic acid in a sample. Where targets are single stranded, probes arecomplementary single strands. Probes can be labeled with reportermolecules for use in hybridization reactions including Southern,northern, in situ, dot blot, array, and like technologies or inscreening assays.

“Protein” refers to a polypeptide or any portion thereof. A “portion” ofa protein refers to that length of amino acid sequence which wouldretain at least one biological activity, a domain identified by PFAM orPRINTS analysis or an antigenic epitope of the protein identified usingKyte-Doolittle algorithms of the PROTEAN program (DNASTAR). An“oligopeptide” is an amino acid sequence from about five residues toabout 15 residues that is used as part of a fusion protein to produce anantibody.

“Purified” refers to any molecule or compound that is separated from itsnatural environment and is from about 60% free to about 90% free fromother components with which it is naturally associated.

“Sample” is used in its broadest sense as containing nucleic acids,proteins, antibodies, and the like. A sample may comprise a bodilyfluid; the soluble fraction of a cell preparation, or an aliquot ofmedia in which cells were grown; a chromosome, an organelle, or membraneisolated or extracted from a cell; genomic DNA, RNA, or cDNA in solutionor bound to a substrate; a cell; a tissue; a tissue print; afingerprint, buccal cells, skin, or hair; and the like.

“Specific binding” refers to a special and precise interaction betweentwo molecules which is dependent upon their structure, particularlytheir molecular side groups. For example, the intercalation of aregulatory protein into the major groove of a DNA molecule, the hydrogenbonding along the backbone between two single stranded nucleic acids, orthe binding between an epitope of a protein and an agonist, antagonist,or antibody.

“Similarity” as applied to sequences, refers to the quantification(usually percentage) of nucleotide or residue matches between at leasttwo sequences aligned using a standardized algorithm such as aSmith-Waterman alignment (Smith and Waterman (1981) J Mol Biol147:195-197) or BLAST2 (Altschul et al. (1997) Nucleic Acids Res25:3389-3402). BLAST2 may be used in a standardized and reproducible wayto insert gaps in one of the sequences in order to optimize alignmentand to achieve a more meaningful comparison between them.

“Substrate” refers to any rigid or semi-rigid support to whichpolynucleotides or proteins are bound and includes membranes, filters,chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels,capillaries or other tubing, plates, polymers, and microparticles with avariety of surface forms including wells, trenches, pins, channels andpores.

“Variant” refers to molecules that are recognized variations of apolynucleotide or a protein encoded by the polynucleotide. Splicevariants may be determined by BLAST score, wherein the score is at least100, and preferably at least 400. Allelic variants have a high percentidentity to the polynucleotides and may differ by about three bases perhundred bases. “Single nucleotide polymorphism” (SNP) refers to a changein a single base as a result of a substitution, insertion or deletion.The change may be conservative (purine for purine) or non-conservative(purine to pyrimidine) and may or may not result in a change in anencoded amino acid

THE INVENTION

The invention is based on the discovery of new human cyclic nucleotidephosphodiesterases (HSPDE10A), the polynucleotides encoding HSPDE10A,and the use of these compositions for the diagnosis and treatment ofcancer and immune disorders.

Nucleic acids encoding the HSPDE10A of the present invention wereidentified in Incyte Clone 826776 from the prostate cDNA library(PROSTUT04) using BLAST analysis and human PDE5 (GI 3355606) as a querysequence against the LIFESEQ database (Incyte Genomics, Palo Alto,Calif.). Full length cDNA sequences of HSPDE10A were obtained from ahuman skeletal muscle library using the complete cDNA insert of IncyteClone 826776 as a hybridization probe. Clone 826776 has identity withthe nucleotide sequence encoding HSPDE10A from nucleotide 627 tonucleotide 888. The oligonucleotide of SEQ ID NO:2 from about nucleotide1168 to about nucleotide 1212 is useful in hybridization oramplification technologies to identify SEQ ID NO:2 and to distinguishbetween SEQ ID NO:2 and a related sequence. The oligonucleotide of SEQID NO:4 from about nucleotide 1183 to about nucleotide 1227 is useful inhybridization or amplification technologies to identify SEQ ID NO:4 andto distinguish between SEQ ID NO:4 and a related sequence. FIG. 7 showsthe electronic northern analysis for HSPDE10A.

In one embodiment, the invention encompasses a protein comprising theamino acid sequence of SEQ ID NO:1, as shown in FIGS. 1A-1E. HSPDE10A1is 490 amino acids in length and has a putative cGMP binding motif inthe sequence N₈₈RLDGKPFDDAD of SEQ ID NO:1 and a PDE signature motif atH₂₆₀DLDHRGTNN of SEQ ID NO:1. As shown in FIGS. 3A-3E, HSPDE10A1 haschemical and structural similarity with human PDE5, HSPDE5A1 (g3355606;SEQ ID NO:5). In particular, HSPDE10A1 and HSPDE5A1 share 42% identity.The ˜270 amino acid catalytic domain found in all PDEs extends betweenresidues F₁₉₆ and R₄₅₈ in HSPDE10A1 and is 50% identical to HSPDE5A1 inthis region. The putative cGMP binding motif in HSPDE10A1 beginning atresidue N₈₈ is coincident with the tandem repeat motif for cGMP bindingin HSPDE5A1 beginning at residue N₄₇₂, and the PDE signature sequencefor HSPDE10A1 beginning at residue H₂₆₀ is conserved in HSPDE5A as well.HSPDE10A1 shares a slightly lesser degree of homology, ranging from 25%to 44%, with other representatives of PDE families 1, 2, 3, 4, 6, 7, 8,and 9 (data not shown). Portions of HSPDE10A which are hydrophillic andappropriate to use as antigenic epitopes include residues 10-25, 45-60,88-103, 145-158, and 260-275. Antibodies produced epitopes comprisingresidues 350-365 and 396-411 would distinguish HSPDE10A1 from HSPDE10A2.These epitopes are synthesized, coupled to KLH and used to produceantibodies which bind specifically to HSPDE10A and are diagnostics forexamining biopsied prostate tissues potentially affected byadenofibromatous hyperplasia.

In another embodiment, the invention encompasses a protein comprisingthe amino acid sequence of SEQ ID NO:3. As shown in FIGS. 2A-2E,HSPDE10A2 is 367 amino acids in length and also contains the putativecGMP binding motif at N₈₈RLDGKPFDDAD of SEQ ID NO:3 and a PDE signaturemotif at H₂₆₀DLDHRGTNN of SEQ ID NO:3. As shown in FIGS. 3A-3E,HSPDE10A2 is identical to HSPDE10A1 between residues M₁ and E₃₃₈, butdiffers in the C-terminal portion of the molecule from E₃₃₉ to Y367.HSPDE10A2 also shares 40% identity with HSPDE5A1.

A cDNA construct encoding the full length amino acid sequence ofHSPDE10A1 was cloned into the baculovirus transfer vector pFASTBAC,expressed in Sf9 cells, and the enzyme partially purified from thesecells for enzyme assays. FIGS. 4A and 4B show the kinetics of HSPDE10A1enzyme activity with cAMP (FIG. 4A) and cGMP (FIG. 4B) as substrates.Both substrates are hydrolyzed at a similar rate (V_(max)=0.23 and 0.21μmole/min/ml enzyme for cAMP and cGMP, respectively), and with a similaraffinity for HSPDE10A1 (K_(m)=1.04 and 0.52 μM for cAMP and cGMP,respectively). The data confirms that HSPDE10A1 is a PDE capable ofhydrolyzing both cAMP and cGMP at physiologically relevantconcentrations. The effects of various known PDE inhibitors on theactivity of HSPDE10A1 using cGMP as a substrate are shown in Table 1.HSPDE10A1 was relatively insensitive to both milrinone and rolipram,which are selective for PDE3 and PDE4 respectively, with IC₅₀ valuesof >200 μM and 160 μM, respectively. The non-selective PDE inhibitorIBMX (3-isobutyl1-methylxanthine) inhibited HSPDE10A1 with an IC₅₀ of 40μM, which is within the range observed for other PDEs, except theIBMX-insensitive PDE8. The so-called cGMP PDE-specific inhibitorzaprinast, which is selective for PDE5 and PDE6, was moderatelyeffectivet against HSPDE10A1 with an IC₅₀ of 8 μM (10-40 fold higherthan PDEs 5 and 6).

The degree of similarity exhibited between the HSPDE10A1 andrepresentatives of the other families of PDEs in the catalytic domain(25% to 50%) is consistent with that demonstrated between different PDEfamilies (˜30%). HSPDE10A1 is further distinguished from other knownfamilies by its dual specificity for both cAMP and cGMP and by itspattern of inhibition by known PDE inhibitors. HSPDE10A1 thereforeappears to be a member of a new family of cyclic nucleotidephosphodiesterases designated PDE10.

Membrane-based northern analysis (FIGS. 5A-5B) shows the expression ofHSPDE10A as a major transcript of ˜7.5 kb in skeletal muscle andprostate tissue. An additional ˜3.0 kb mRNA was detected in prostatealone; a less prominent transcript of ˜1.5 kb occurs in skeletal muscleand testes. These data suggest the existence of at least three HSPDE10Asplice variants. Electronic northern analysis using the LIFESEQ database(Incyte Genomics) further shows the expression of HSPDE10A in cancerousprostate tissue.

FIG. 6 shows the expression of HSPDE10A1 in cell lysates of Sf9 cellstransformed with a baculovirus vector containing an untagged cDNAconstruct. An ˜56 kDa protein was detected by Coomassie blue staining(native HSPDE10A1; FIG. 6) and by western immunoblotting of aFLAG-tagged HSPDE10A1 using an anti-FLAG antibody (data not shown).

The invention also encompasses polynucleotides which encode HSPDE10A. Ina particular embodiment, the invention encompasses a polynucleotidecomprising the nucleic acid sequence of SEQ ID NO:2 which encodesHSPDE10A1. In another embodiment, the invention encompasses apolynucleotide comprising the nucleic acid sequence of SEQ ID NO:4 whichencodes HSPDE10A2.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of polynucleotidesequences encoding HSPDE10A, some bearing minimal similarity to thepolynucleotide sequences of any known and naturally occurring gene, maybe produced. Thus, the invention contemplates each and every possiblevariation of polynucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe polynucleotide sequence of naturally occurring HSPDE10A, and allsuch variations are to be considered as being specifically disclosed.

Although nucleotide sequences which encode HSPDE10A and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring HSPDE10A under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding HSPDE10A or its derivatives possessing a substantiallydifferent codon usage, e.g., inclusion of non-naturally occurringcodons. Codons may be selected to increase the rate at which expressionof the peptide occurs in a particular prokaryotic or eukaryotic host inaccordance with the frequency with which particular codons are utilizedby the host. Other reasons for substantially altering the nucleotidesequence encoding HSPDE10A and its derivatives without altering theencoded amino acid sequences include the production of RNA transcriptshaving more desirable properties, such as a greater half-life, thantranscripts produced from the naturally occurring sequence.

The invention also encompasses production of DNA sequences which encodeHSPDE10A and HSPDE10A derivatives, or fragments thereof, entirely bysynthetic chemistry. After production, the synthetic sequence may beinserted into any of the many available expression vectors and cellsystems using reagents well known in the art. Moreover, syntheticchemistry may be used to introduce mutations into a sequence encodingHSPDE10A.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed polynucleotide sequences, and, inparticular, to those shown in SEQ ID NO:2, or to a fragment of SEQ IDNO:2, under various conditions of stringency (Wahl and Berger (1987)Methods Enzymol 152:399-407; Kimmel (1987) Methods Enzymol 152:507-511).For example, stringent salt concentration will ordinarily be less thanabout 750 mM NaCl and 75 mM trisodium citrate (SSC), preferably lessthan about 500 mM NaCl and 50 mM SSC, and most preferably less thanabout 250 mM NaCl and 25 mM SSC. Low stringency hybridization can beobtained in the absence of organic solvent, e.g., formamide, while highstringency hybridization can be obtained in the presence of at leastabout 35% formamide, and most preferably at least about 50% formamide.Stringent temperature conditions will ordinarily include temperatures ofat least about 30° C., more preferably of at least about 37° C., andmost preferably of at least about 42° C. Varying additional parameters,such as hybridization time, the concentration of detergent, e.g., sodiumdodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA,are well known to those skilled in the art. Various levels of stringencyare accomplished by combining these various conditions as needed. In apreferred embodiment, hybridization will occur at 30° C. in 750 mM NaCl,75 mM SSC, and 1% SDS. In a more preferred embodiment, hybridizationwill occur at 37° C. in 500 mM NaCl, 50 mM SSC, 1% SDS, 35% formamide,and 100 μg/ml denatured salmon sperm DNA (ssDNA). In a most preferredembodiment, hybridization will occur at 42° C. in 250 mM NaCl, 25 mMSSC, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations onthese conditions will be readily apparent to those skilled in the art.

The washing steps which follow hybridization can also vary instringency. Wash stringency conditions can be defined by saltconcentration and by temperature. As above, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentration for the wash steps willpreferably be less than about 30 mM NaCl and 3 mM SSC, and mostpreferably less than about 15 mM NaCl and 1.5 mM SSC. Stringenttemperature conditions for the wash steps will ordinarily includetemperature of at least about 25° C., more preferably of at least about42° C., and most preferably of at least about 68° C. In a preferredembodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3 mM SSC, and0.1% SDS. In a more preferred embodiment, wash steps will occur at 42°C. in 15 mM NaCl, 1.5 mM SSC, and 0.1% SDS. In a most preferredembodiment, wash steps will occur at 68° C. in 15 mM NaCl, 1.5 mM SSC,and 0.1% SDS. Additional variations on these conditions will be readilyapparent to those skilled in the art.

Methods for DNA sequencing are well known in the art and may be used topractice any of the embodiments of the invention. The methods may employsuch enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE, Taqpolymerase, thermostable T7 polymerase (Amersham Pharmacia Biotech(APB), Piscataway, N.J.), or combinations of polymerases andproofreading exonucleases such as those found in the ELONGASEamplification system (Life Technologies, Gaithersburg, Md.). Preferably,sequence preparation is automated with machines such as the RobbinsHydra microdispenser (Robbins Scientific, Sunnyvale, Calif.), MICROLAB2200 system (Hamilton, Reno, Nev.), DNA ENGINE thermal cycler (MJResearch, Watertown, Mass.) and the ABI CATALYST 800 thermal cycler (PEBiosystems). Sequencing is then carried out using either ABI 373 or 377DNA sequencing systems (PE Biosystems) or the MEGABACE 1000 DNAsequencing system (APB). The resulting sequences are analyzed using avariety of algorithms which are well known in the art and reviewed inAusubel (1997; Short Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y., unit 7.7) and in Meyers (1995; Molecular Biology andBiotechnology, Wiley VCH, New York, N.Y., pp. 856-853).

The nucleic acid sequences encoding HSPDE10A may be extended utilizing apartial nucleotide sequence and employing various PCR-based methodsknown in the art to detect upstream sequences, such as promoters andregulatory elements. For example, one method which may be employed,restriction-site PCR, uses universal and nested primers to amplifyunknown sequence from genomic DNA within a cloning vector. (See, e.g.,Sarkar (1993) PCR Methods Applic 2:318-322.) Another method, inversePCR, uses primers that extend in divergent directions to amplify unknownsequence from a circularized template. The template is derived fromrestriction fragments comprising a known genomic locus and surroundingsequences. (See, e.g., Triglia et al. (1988) Nucleic Acids Res 16:8186.)A third method, capture PCR, involves PCR amplification of DNA fragmentsadjacent to known sequences in human and yeast artificial chromosomeDNA. (See, e.g., Lagerstrom et al. (1991) PCR Methods Applic 1:111-119.)In this method, multiple restriction enzyme digestions and ligations maybe used to insert an engineered double-stranded sequence into a regionof unknown sequence before performing PCR. Other methods which may beused to retrieve unknown sequences are known in the art. (See, e.g.,Parker et al. (1991) Nucleic Acids Res 19:3055-306). Additionally, onemay use PCR, nested primers, and PROMOTERFINDER libraries (Clontech,Palo Alto, Calif.) to walk genomic DNA. This procedure avoids the needto screen libraries and is useful in finding intron/exon junctions. Forall PCR-based methods, primers may be designed using commerciallyavailable software, such as OLIGO 4.06 primer analysis software(National Biosciences, Plymouth, Minn.) or another appropriate program,to be about 22 to 30 nucleotides in length, to have a GC content ofabout 50% or more, and to anneal to the template at temperatures ofabout 68° C. to 72° C. When screening for full-length cDNAs, it ispreferable to use libraries that have been size-selected to includelarger cDNAs. In addition, random-primed libraries, which often includesequences containing the 5′ regions of genes, are preferable forsituations in which an oligo d(T) library does not yield a full-lengthcDNA. Genomic libraries may be useful for extension of sequence into 5′non-transcribed regulatory regions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentnucleotide-specific, laser-stimulated fluorescent dyes, and a chargecoupled device camera for detection of the emitted wavelengths.Output/light intensity may be converted to electrical signal usingGENOTYPER or SEQUENCE NAVIGATOR software (PE Biosystems), and the entireprocess from loading of samples to computer analysis and electronic datadisplay may be computer controlled. Capillary electrophoresis isespecially preferable for sequencing small DNA fragments which may bepresent in limited amounts in a particular sample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode HSPDE10A may be cloned in recombinant DNAmolecules that direct expression of HSPDE10A, or fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and used to express HSPDE10A.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alterHSPDE10A-encoding sequences for a variety of purposes including, but notlimited to, modification of the cloning, processing, and/or expressionof the gene product. DNA shuffling by random fragmentation and PCRreassembly of gene fragments and synthetic oligonucleotides may be usedto engineer the nucleotide sequences. For example,oligonucleotide-mediated site-directed mutagenesis may be used tointroduce mutations that create new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, and so forth.

In another embodiment, sequences encoding HSPDE10A may be synthesized,in whole or in part, using chemical methods well known in the art anddescribed by Caruthers et al. (1980; Nucleic Acids Symp Ser (7) 215-223)and Horn et al. (1980; Nucleic Acids Symp Ser (7) 225-232).Alternatively, HSPDE10A itself or a fragment thereof may be synthesizedusing chemical methods. For example, peptide synthesis can be performedusing various solid-phase techniques(Roberge et al. (1995) Science269:202-204). Automated synthesis may be achieved using the ABI 431Apeptide synthesizer (PE Biosystems). Additionally, the amino acidsequence of HSPDE10A, or any part thereof, may be altered during directsynthesis and/or combined with sequences from other proteins, or anypart thereof, to produce a variant protein.

The peptide may be substantially purified by preparative highperformance liquid chromatography. (Chiez and Regnier (1990) MethodsEnzymol 182:392-421). The composition of the synthetic peptides may beconfirmed by amino acid analysis or by sequencing. (See, e.g., Creighton(1984) Proteins, Structures and Molecular Properties, W H Freeman, NewYork, N.Y.)

In order to express a biologically active HSPDE10A, the nucleotidesequences encoding HSPDE10A or derivatives thereof may be inserted intoan appropriate expression vector, i.e., a vector which contains thenecessary elements for transcriptional and translational control of theinserted coding sequence in a host. These elements include regulatorysequences, such as enhancers, constitutive and inducible promoters, and5′ and 3′ untranslated regions in the vector and in polynucleotidesequences encoding HSPDE10A. Such elements may vary in their strengthand specificity. Specific initiation signals may also be used to achievemore efficient translation of sequences encoding HSPDE10A. Such signalsinclude the ATG initiation codon and adjacent sequences, e.g. the Kozaksequence. In cases where sequences encoding HSPDE10A and its initiationcodon and upstream regulatory sequences are inserted into theappropriate expression vector, no additional transcriptional ortranslational control signals may be needed. However, in cases whereonly coding sequence, or a fragment thereof, is inserted, exogenoustranslational control signals including an in-frame ATG initiation codonshould be provided by the vector. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers appropriate for the particular host cell system used (Scharfet al. (1994) Results Probl Cell Differ 20:125-162).

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding HSPDE10A andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination as described in Sambrooket al. (1989; Molecular Cloning, A Laboratory Manual, Cold Spring HarborPress, Plainview, N.Y., ch. 4, 8, and 16-17) or in Ausubel et al. (1995;Current Protocols in Molecular Biology, John Wiley & Sons, New York,N.Y., ch. 9, 13, and 16).

A variety of expression vector/host systems may be utilized to containand express sequences encoding HSPDE10A. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith baculovirus expression vectors; plant cell systems transformed withviral expression vectors (cauliflower mosaic virus, CaMV, or tobaccomosaic virus, TMV) or with bacterial expression vectors (Ti or pBR322plasmids); or animal cell systems. The invention is not limited by thehost cell employed.

In bacterial systems, a number of cloning and expression vectors may beselected depending upon the use intended for polynucleotide sequencesencoding HSPDE10A. For example, routine cloning, subcloning, andpropagation of polynucleotide sequences encoding HSPDE10A can beachieved using a multifunctional E. coli vector such as PBLUESCRIPT(Stratagene, La Jolla Calif.) or PSPORT1 plasmid (Life Technologies).Ligation of sequences encoding HSPDE10A into the vector's multiplecloning site disrupts the lacZ gene, allowing a colorimetric screeningprocedure for identification of transformed bacteria containingrecombinant molecules. In addition, these vectors may be useful for invitro transcription, dideoxy sequencing, single strand rescue withhelper phage, and creation of nested deletions in the cloned sequence(Van Heeke and Schuster (1989) J Biol Chem 264:5503-5509). When largequantities of protein are needed for the production of antibodies,vectors containing the strong, inducible T5 or T7 bacteriophage promoterwhich direct high level expression of may be used.

Yeast expression systems may be used for production of HSPDE10A. Anumber of vectors containing constitutive or inducible promoters, suchas alpha factor, alcohol oxidase, and PGH promoters, may be used in theyeast Saccharomyces cerevisiae or Pichia pastoris. In addition, suchvectors direct either the secretion or intracellular retention ofexpressed proteins and enable integration of foreign sequences into thehost genome for stable propagation. (See, e.g., Ausubel (1995) supra;Grant et al. (1987) Methods Enzymol 153:516-54; and Scorer et al. (1994)Biotechnol 12:181-184.)

Plant systems may also be used for expression of HSPDE10A. Transcriptionof sequences encoding HSPDE10A may be driven by viral promoters such asthe 35S and 19S promoters of CaMV used alone or in combination with theomega leader sequence from TMV (Takamatsu (1987) EMBO J 6:307-311).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi et al. (1984) EMBO J3:1671-1680; Broglie et al. (1984) Science 224:838-843; and Winter etal. (1991) Results Probl Cell Differ 17:85-105). These constructs can beintroduced into plant cells by direct DNA or pathogen-mediatedtransformation (The McGraw Hill Yearbook of Science and Technology(1992) McGraw Hill, New York, N.Y., pp. 191-196).

In mammalian cells, a number of viral-based expression systems may beutilized. In cases where an adenovirus is used as an expression vector,sequences encoding HSPDE10A may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain infective virus whichexpresses HSPDE10A in host cells (Logan and Shenk (1984) Proc Natl AcadSci 81:3655-3659). In addition, transcription enhancers, such as the RSVenhancer, may be used to increase expression in mammalian host cells.SV40 or EBV-based vectors may also be used for high-level proteinexpression.

Human artificial chromosomes (HACs) may also be employed to deliverlarger fragments of DNA than can be contained in and expressed from aplasmid. HACs of about 6 kb to 10 Mb are constructed and delivered viaconventional delivery methods (liposomes, polycationic amino polymers,or vesicles) for therapeutic purposes (Harrington et al. (1997) NatureGenet 15:345-355).

For long term production of recombinant proteins in mammalian systems,stable expression of HSPDE10A in cell lines is preferred. For example,sequences encoding HSPDE10A can be transformed into cell lines usingexpression vectors which may contain viral origins of replication and/orendogenous expression elements and a selectable marker gene on the sameor on a separate vector. Following the introduction of the vector, cellsmay be allowed to grow for about 1 to 2 days in enriched media beforebeing switched to selective media. The purpose of the selectable markeris to confer resistance to a selective agent, and its presence allowsgrowth and recovery of cells which successfully express the introducedsequences. Resistant clones of stably transformed cells may bepropagated using tissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase and adenine phosphoribosyltransferase genes, for use intk⁻ or apr⁻cells, respectively (Wigler et al. (1977) Cell 11:223-232;Lowy et al. (1980) Cell 22:817-823). Also, antimetabolite, antibiotic,or herbicide resistance can be used as the basis for selection. Forexample, dhfr confers resistance to methotrexate; neo confers resistanceto the aminoglycosides, neomycin and G-418; and als or pat conferresistance to chlorsulfuron and phosphinotricin acetyltransferase,respectively (Wigler et al. (1980) Proc Natl Acad Sci 77:3567-3570;Colbere-Garapin et al. (1981) J Mol Biol 150:1-14). Additionalselectable genes have been described, trpB and hisD, which altercellular requirements for metabolites (Hartman and Mulligan (1988) ProcNatl Acad Sci 85:8047-8051). Visible markers, e.g., anthocyanins, greenfluorescent proteins (GFP; Clontech), β glucuronidase and its substrateβ-glucuronide, or luciferase and its substrate luciferin may be used.These markers can be used not only to identify transformants, but alsoto quantify the amount of transient or stable expression attributable toa specific vector system (Rhodes (1995) Methods Mol Biol 55:121-131).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, the presence and expression of thegene may need to be confirmed. For example, if the sequence encodingHSPDE10A is inserted within a marker gene sequence, transformed cellscontaining sequences encoding HSPDE10A can be identified by the absenceof marker gene function. Alternatively, a marker gene can be placed intandem with a sequence encoding HSPDE10A under the control of a singlepromoter. Expression of the marker gene in response to induction orselection usually indicates expression of the tandem gene as well.

In general, host cells that contain the nucleic acid sequence encodingHSPDE10A and that express HSPDE10A may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCRamplification, and protein bioassay or immunoassay techniques whichinclude membrane, solution, or chip based technologies for the detectionand/or quantification of nucleic acid sequences.

Immunological methods for detecting and measuring the expression ofHSPDE10A using either specific polyclonal or monoclonal antibodies areknown in the art. Examples of such techniques include enzyme-linkedimmunosorbent assays (ELISAs), radioimmunoassays (RIAs), andfluorescence activated cell sorting (FACS). A two-site, monoclonal-basedimmunoassay utilizing monoclonal antibodies reactive to twonon-interfering epitopes on HSPDE10A is preferred, but a competitivebinding assay may be employed. These and other assays are well known inthe art (Hampton et al. (1990) Serological Methods, a Laboratory Manual,APS Press, St Paul, Minn., Sect. IV; Coligan et al. (1997) CurrentProtocols in Immunology, Greene Pub. Associates and Wiley-Interscience,New York, N.Y.; and Pound (1998) Immunochemical Protocols, Humana Press,Totowa, N.J.).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding HSPDE10A includeoligolabeling, nick translation, end-labeling, or PCR amplificationusing a labeled nucleotide. Alternatively, the sequences encodingHSPDE10A, or any fragments thereof, may be cloned into a vector for theproduction of an mRNA probe. Such vectors are known in the art, arecommercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as T7, T3, orSP6 and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits, such as those provided by APBand Promega (Madison, Wis.). Reporter molecules or labels which may beused for ease of detection include radionuclides, enzymes, fluorescent,chemiluminescent, or chromogenic agents, as well as substrates,cofactors, inhibitors, magnetic particles, and the like.

Host cells transformed with nucleotide sequences encoding HSPDE10A maybe cultured under conditions for the expression and recovery of theprotein from cell culture. The protein produced by a transformed cellmay be secreted or retained intracellularly depending on the sequenceand/or the vector used. As will be understood by those of skill in theart, expression vectors containing polynucleotides which encode HSPDE10Amay be designed to contain signal sequences which direct secretion ofHSPDE10A through a prokaryotic or eukaryotic cell membrane.

In addition, a host cell strain may be chosen for its ability tomodulate expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of theprotein include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to specify protein targeting, folding, and/oractivity. Different host cells which have specific cellular machineryand characteristic mechanisms for post-translational activities (CHO,HeLa, MDCK, HEK293, WI38 and the like) are available from the ATCC(Manassas, Va.) and may be chosen to ensure the correct modification andprocessing of the recombinant protein.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding HSPDE10A may be ligated to aheterologous sequence resulting in translation of a fusion protein inany of the aforementioned host systems. For example, a chimeric HSPDE10Aprotein containing a heterologous moiety that can be recognized by acommercially available antibody may facilitate the screening of peptidelibraries for inhibitors of HSPDE10A activity. Heterologous protein andpeptide moieties may also facilitate purification of fusion proteinsusing commercially available affinity matrices. Such moieties include,but are not limited to, glutathione S-transferase (GST), maltose bindingprotein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP),6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and6-His enable purification of their cognate fusion proteins onimmobilized glutathione, maltose, phenylarsine oxide, calmodulin, andmetal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA)enable immunoaffinity purification of fusion proteins using commerciallyavailable monoclonal and polyclonal antibodies that specificallyrecognize these epitope tags. A fusion protein may also be engineered tocontain a proteolytic cleavage site located between the HSPDE10Aencoding sequence and the heterologous protein sequence, so thatHSPDE10A may be cleaved away from the heterologous moiety followingpurification. Methods for fusion protein expression and purification arediscussed in Ausubel (1995, supra, ch 10). A variety of commerciallyavailable kits may also be used to facilitate expression andpurification of fusion proteins.

In a further embodiment of the invention, synthesis of radiolabeledHSPDE10A may be achieved in vitro using the TNT rabbit reticulocytelysate or wheat germ extract systems (Promega). These systems coupletranscription and translation of protein-coding sequences operablyassociated with the T7, T3, or SP6 promoters. Translation takes place inthe presence of a radiolabeled amino acid precursor, preferably³⁵S-methionine.

Fragments of HSPDE10A may be produced not only by recombinantproduction, but also by direct peptide synthesis using solid-phasetechniques (Creighton, supra, pp. 55-60). Protein synthesis may beperformed by manual techniques or by automation. Automated synthesis maybe achieved, for example, using the ABI 431A peptide synthesizer (PEBiosystems). Various fragments of HSPDE10A may be synthesized separatelyand then combined to produce the full length molecule.

THERAPEUTICS

Chemical and structural similarity in the context of sequences andmotifs exists between regions of HSPDE10A and human cyclic nucleotidephosphodiesterases. In addition, the expression of HSPDE10A is closelyassociated with normal skeletal muscle and prostate tissues. Therefore,HSPDE10A appears to be downregulated in prostate cancer, specifically inadenofibromatous hyperplasia. In the treatment of this and otherdisorders associated with decreased HSPDE10A expression or activity, itis desirable to increase the expression or activity of HSPDE10A bydelivery of the protein, a vector expressing the protein or an agonistof HSPDE10A. In those disorders in which the increased expression of theprotein is implicated in the cancerous or immune disease process, it isdesirable to decrease expression or activity of HSPDE10A by delivery ofan antibody, antagonist, or inhibitor.

Therefore, in one embodiment, HSPDE10A or a fragment or derivativethereof may be administered to a subject to treat a disorder associatedwith decreased expression or activity of HSPDE10A. Examples of suchdisorders include, but are not limited to, a cancer, such as cancer suchas adenocarcinoma, melanoma, myeloma, sarcoma, and teratocarcinoma ofthe bone, bone marrow, ganglia, gastrointestinal tract, heart, kidney,liver, lung, muscle, nerve, prostate, testis, and uterus and an immunedisorder such as adult respiratory distress syndrome, asthma,atherosclerosis, cholecystitis, Crohn's disease, dermatomyositis,emphysema, erythema nodosum, gastritis, glomerulonephritis, multiplesclerosis, myasthenia gravis, myopathies, osteoarthritis, osteoporosis,polymyositis, rheumatoid arthritis, scleroderma, systemic lupuserythematosus, systemic sclerosis, and ulcerative colitis.

In a second embodiment, a vector capable of expressing HSPDE10A or afragment or derivative thereof may be administered to a subject to treata disorder associated with decreased expression or activity of HSPDE10Aincluding, but not limited to, those described above.

In a third embodiment, a composition comprising a substantially purifiedHSPDE10A in conjunction with a carrier may be administered to a subjectto treat a disorder associated with decreased expression or activity ofHSPDE10A including, but not limited to, those provided above.

In a fourth embodiment, a composition comprising an agonist whichspecifically binds HSPDE10A and which increases the expression,activity, or lifespan of HSPDE10A may be administered to a subject totreat a disorder associated with decreased expression or activity ofHSPDE10A including, but not limited to, those listed above.

In a fifth embodiment, an antagonist of HSPDE10A may be administered toa subject to treat or prevent a disorder associated with increasedexpression or activity of HSPDE10A. Such disorders may include, but arenot limited to, those discussed above. In one aspect, an antibody whichspecifically binds HSPDE10A may be used directly as an antagonist orindirectly as a targeting or delivery mechanism for bringing apharmaceutical agent to cells or tissue which express HSPDE10A.

In a sixth embodiment, a vector expressing the complement of thepolynucleotide encoding HSPDE10A may be administered to a subject totreat a disorder associated with increased expression or activity ofHSPDE10A including, but not limited to, those described above.

In other embodiments, any of the proteins, antagonists, antibodies,agonists, complementary sequences, or vectors of the invention may beadministered in combination with other appropriate therapeutic agents.Selection of the appropriate agents for use in combination therapy maybe made by one of ordinary skill in the art, according to conventionalpharmaceutical principles. The combination of therapeutic agents may actsynergistically to effect the treatment of the various disordersdescribed above. Using this approach, one may be able to achievetherapeutic efficacy with lower dosages of each agent, thus reducing thepotential for adverse side effects.

An antagonist of HSPDE10A may be produced using methods which aregenerally known in the art. In particular, purified HSPDE10A may be usedto produce antibodies or to screen libraries of pharmaceutical agents toidentify those which specifically bind HSPDE10A. Antibodies to HSPDE10Amay also be generated using methods that are well known in the art. Suchantibodies may include, but are not limited to, polyclonal, monoclonal,chimeric, and single chain antibodies, Fab fragments, and fragmentsproduced by a Fab expression library. Neutralizing antibodies areespecially preferred for therapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others may be immunized by injectionwith HSPDE10A or with any fragment or oligopeptide thereof which hasimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase immunological response. Such adjuvants include,but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) andCorynebacterium parvum are especially preferable.

It is preferred that oligopeptides having from about 5 to about 15 aminoacids and selected from the antigenic regions of SEQ ID NOs:1 and 3 areused to induce antibodies to the proteins. These oligopeptides which areidentical to epitopes of the natural protein may be fused with those ofanother protein, such as KLH, and antibodies produced to the chimericmolecule.

Monoclonal antibodies to HSPDE10A may be prepared using any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique (Kohler et al. (1975) Nature 256:495-497; Kozboret al. (1985) J Immunol Methods 81:31-42; Cote et al. (1983) Proc NatlAcad Sci 80:2026-2030; and Cole et al. (1984) Mol Cell Biol 62:109-120).

In addition, techniques developed for the production of “chimericantibodies,” such as the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity, can be used (Morrison et al. (1984) Proc NatlAcad Sci 81:6851-6855; Neuberger et al. (1984) Nature 312:604-608; andTakeda et al. (1985) Nature 314:452-454). Alternatively, techniquesdescribed for the production of single chain antibodies may be adapted,using methods known in the art, to produce HSPDE10A-specific singlechain antibodies. Antibodies with related specificity, but of distinctidiotypic composition, may be generated by chain shuffling from randomcombinatorial immunoglobulin libraries (Burton (1991) Proc Natl Acad Sci88:10134-10137).

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature(Orlandi et al. (1989) Proc Natl Acad Sci 86: 3833-3837; Winter et al.(1991) Nature 349:293-299).

Antibody fragments which contain specific binding sites for HSPDE10A mayalso be generated. For example, such fragments include, but are notlimited to, F(ab′)2 fragments produced by pepsin digestion of theantibody molecule and Fab fragments generated by reducing the disulfidebridges of the F(ab′)2 fragments. Alternatively, Fab expressionlibraries may be constructed to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity (Huse et al.(1989) Science 246:1275-1281).

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between HSPDE10A and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering HSPDE10A epitopes is preferred, but a competitivebinding assay may also be employed (Pound, supra).

Various methods such as Scatchard analysis in conjunction withradioimmunoassay techniques may be used to assess the affinity ofantibodies for HSPDE10A. Affinity is expressed as an associationconstant, K_(a), which is defined as the molar concentration ofHSPDE10A-antibody complex divided by the molar concentrations of freeantigen and free antibody under equilibrium conditions. The K_(a)determined for a preparation of polyclonal antibodies, which areheterogeneous in their affinities for multiple HSPDE10A epitopes,represents the average affinity, or avidity, of the antibodies forHSPDE10A. The K_(a) determined for a preparation of monoclonalantibodies, which are monospecific for a particular HSPDE10A epitope,represents a true measure of affinity. High-affinity antibodypreparations with K_(a) ranging from about 10⁹ to 10¹² l/mole arepreferred for use in immunoassays in which the HSPDE10A-antibody complexmust withstand rigorous manipulations. Low-affinity antibodypreparations with K_(a) ranging from about 10⁶ to 10⁷ l/mole arepreferred for use in immunopurification and similar procedures whichultimately require dissociation of HSPDE10A, preferably in active form,from the antibody (Catty (1988) Antibodies, Volume I: A PracticalApproach, IRL Press, Washington, D.C.; Liddell and Cryer (1991) APractical Guide to Monoclonal Antibodies, John Wiley & Sons, New York,N.Y.).

The titer and avidity of polyclonal antibody preparations may be furtherevaluated to determine the quality and suitability of such preparationsfor certain downstream applications. For example, a polyclonal antibodypreparation containing at least 1-2 mg specific antibody/ml, preferably5-10 mg specific antibody/ml, is preferred for use in proceduresrequiring precipitation of HSPDE10A-antibody complexes. Procedures forevaluating antibody specificity, titer, and avidity, and guidelines forantibody quality and usage in various applications, are generallyavailable. (See, e.g., Catty, supra, and Coligan, supra.)

In another embodiment of the invention, the polynucleotides encodingHSPDE10A, or any fragment or complement thereof, may be used fortherapeutic purposes. In one aspect, the complement of thepolynucleotide encoding HSPDE10A may be used in situations in which itwould be desirable to block the transcription of the mRNA. Inparticular, cells may be transformed with sequences complementary topolynucleotides encoding HSPDE10A. Thus, complementary molecules orfragments may be used to modulate HSPDE10A activity, or to achieveregulation of gene function. Such technology is now well known in theart, and sense or antisense oligonucleotides or larger fragments can bedesigned from various locations along the coding or control regions ofsequences encoding HSPDE10A.

Expression vectors derived from retroviruses, adenoviruses, or herpes orvaccinia viruses, or from various bacterial plasmids, may be used fordelivery of nucleotide sequences to the targeted organ, tissue, or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct vectors to express nucleic acid sequencescomplementary to the polynucleotides encoding HSPDE10A. (See, e.g.,Sambrook, supra; Ausubel, 1995, supra.)

Genes encoding HSPDE10A can be turned off by transforming a cell ortissue with expression vectors which express high levels of apolynucleotide, or fragment thereof, encoding HSPDE10A. Such constructsmay be used to introduce untranslatable sense or antisense sequencesinto a cell. Even in the absence of integration into the DNA, suchvectors may continue to transcribe RNA molecules until they are disabledby endogenous nucleases. Transient expression may last for a month ormore with a non-replicating vector, and may last even longer ifappropriate replication elements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning complementary sequences or antisense molecules (DNA, RNA, orPNA) to the control, 5′, or regulatory regions of the gene encodingHSPDE10A. Oligonucleotides derived from the transcription initiationsite, e.g., between about positions −10 and +10 from the start site, arepreferred. Similarly, inhibition can be achieved using triple helixbase-pairing methodology. Triple helix pairing is useful because itcauses inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described by Gee et al. (1994; In: Huber and Carr, Molecular andImmunologic Approaches, Futura Publishing, Mt. Kisco, N.Y., pp.163-177). A complementary sequence or antisense molecule may also bedesigned to block translation of mRNA by preventing the transcript frombinding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Forexample, engineered hammerhead motif ribozyme molecules may specificallyand efficiently catalyze endonucleolytic cleavage of sequences encodingHSPDE10A.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites, including the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides, corresponding to the region of the target genecontaining the cleavage site, may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes of the inventionmay be prepared by any method known in the art for the synthesis ofnucleic acid molecules. These include techniques for chemicallysynthesizing oligonucleotides such as solid phase phosphoramiditechemical synthesis. Alternatively, RNA molecules may be generated by invitro and in vivo transcription of DNA sequences encoding HSPDE10A. SuchDNA sequences may be incorporated into a wide variety of vectors withRNA polymerase promoters such as T7 or SP6. Alternatively, these cDNAconstructs that synthesize complementary RNA, constitutively orinducibly, can be introduced into cell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5′ and/or 3′ ends of the molecule,or the use of phosphorothioate or 2′O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availablefor use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors maybe introduced into stem cells taken from the patient and clonallypropagated for autologous transplant back into that same patient.Delivery by transfection, by liposome injections, or by polycationicamino polymers may be achieved using methods described by Goldman et al.(1997) Nature Biotechnol 15:462-466).

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical or sterile composition, in conjunction with apharmaceutically acceptable carrier, for any of the therapeutic effectsdiscussed above. Such compositions may consist of HSPDE10A, antibodiesto HSPDE10A, and mimetics, agonists, antagonists, or inhibitors ofHSPDE10A. The compositions may be administered alone or, which may beadministered in any. The compositions may be administered to a patientalone, with a stabilizing compound, with a sterile, biocompatiblecarrier such as saline, buffered saline, dextrose, or water and incombination with other agents, drugs, or hormones.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

In addition to the active ingredients, these compositions may compriseexcipients and auxiliaries which facilitate processing of the activecompounds. Further details on techniques for formulation andadministration may be found in the latest edition of Remington'sPharmaceutical Sciences (Maack Publishing, Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages for oral administration. Such carriers enable the pharmaceuticalcompositions to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions, and the like, foringestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombining active compounds with solid excipient and processing theresultant mixture of granules (optionally, after grinding) to obtaintablets or dragee cores. Auxiliaries can be added, if desired.Excipients include carbohydrate or protein fillers, such as sugars,including lactose, sucrose, mannitol, and sorbitol; starch from corn,wheat, rice, potato, or other plants; cellulose, such as methylcellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums, including arabic and tragacanth; andproteins, such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, and alginic acid or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and organic solvents or solvent mixtures.Dyestuffs or pigments may be added to the tablets or dragee coatings forproduct identification or to characterize the quantity of activecompound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with fillers or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in liquids, such as fatty oils, liquid, orliquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations for parenteral administration may beformulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks'solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Lipophilic solvents or vehicles include fattyoils, such as sesame oil, or synthetic fatty acid esters, such as ethyloleate, triglycerides, or liposomes. Non-lipid polycationic aminopolymers may also be used for delivery. Optionally, the suspension mayalso contain stabilizers or agents to increase the solubility of thecompounds and allow for the preparation of highly concentratedsolutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, and succinic acid. Salts tendto be more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2% to 7%mannitol, at a pH range of 4.5 to 5.5, that is combined with bufferprior to use.

Pharmaceutical compositions for use in the invention includecompositions wherein the determination of an effective dose is wellwithin the capability of those skilled in the art.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells or inanimal models such as mice, rats, rabbits, dogs, or pigs. An animalmodel may also be used to determine the appropriate concentration rangeand route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example HSPDE10A or fragments thereof, antibodies ofHSPDE10A, and agonists, antagonists or inhibitors of HSPDE10A, whichameliorates the symptoms or condition. Therapeutic efficacy and toxicitymay be determined by standard pharmaceutical procedures in cell culturesor with experimental animals, such as by calculating the ED₅₀ (the dosetherapeutically effective in 50% of the population) or LD₅₀ (the doselethal to 50% of the population) statistics. The dose ratio of toxic totherapeutic effects is the therapeutic index, and it can be expressed asthe LD₅₀/ED₅₀ ratio. Pharmaceutical compositions which exhibit largetherapeutic indices are preferred. The data obtained from cell cultureassays and animal studies are used to formulate a range of dosage forhuman use. The dosage contained in such compositions is preferablywithin a range of circulating concentrations that includes the ED₅₀ withlittle or no toxicity. The dosage varies within this range dependingupon the dosage form employed, the sensitivity of the patient, and theroute of administration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject requiring treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, the generalhealth of the subject, the age, weight, and gender of the subject, timeand frequency of administration, drug combination(s), reactionsensitivities, and response to therapy. Long-acting pharmaceuticalcompositions may be administered every 3 to 4 days, every week, orbiweekly depending on the half-life and clearance rate of the particularformulation.

Normal dosage amounts may vary from about 0.1 μg to 100,000 μg, up to atotal dose of about 1 gram, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or proteins will be specific to particular cells,conditions, locations, etc.

DIAGNOSTICS

In another embodiment, antibodies which specifically bind HSPDE10A maybe used for the diagnosis of disorders such as adenofibromatoushyperplasia of the prostate which is characterized by differentialexpression of HSPDE10A or in assays to monitor patients being treatedwith HSPDE10A or agonists, antagonists, or inhibitors of HSPDE10A.Antibodies useful for diagnostic purposes may be prepared in the samemanner as described above for therapeutics. Diagnostic assays forHSPDE10A include methods which utilize the antibody and a label todetect HSPDE10A in human body fluids or in extracts of cells or tissues.The antibodies may be used with or without modification, and may belabeled by covalent or non-covalent attachment of a reporter molecule. Awide variety of reporter molecules, several of which are describedabove, are known in the art and may be used.

A variety of protocols for measuring HSPDE10A, including ELISAs, RIAsand FACS, are known in the art and provide a basis for diagnosingaltered or abnormal levels of HSPDE10A expression. Normal or standardvalues for HSPDE10A expression are established by combining body fluidsor cell extracts taken from normal mammalian subjects, preferably human,with antibody to HSPDE10A under conditions for complex formation. Theamount of standard complex formation may be quantitated by variousmethods, preferably by photometric means. Quantities of HSPDE10Aexpressed in subject, control, and disease samples from biopsied tissuesare compared with the standard values. Deviation between standard andsubject values establishes the parameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encodingHSPDE10A may be used for diagnostic purposes. The polynucleotides whichmay be used include oligonucleotide sequences, complementary RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofHSPDE10A may be correlated with disease. The diagnostic assay may beused to determine absence, presence, and excess expression of HSPDE10A,and to monitor regulation of HSPDE10A levels during therapeuticintervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding HSPDE10A or closely related molecules may be used to identifynucleic acid sequences which encode HSPDE10A. The specificity of theprobe, whether it is made from a highly specific region, such as the 5′regulatory region, or from a less specific region, such as a conservedmotif, and the stringency of the hybridization or amplification(maximal, high, intermediate, or low), will determine whether the probeidentifies only naturally occurring sequences encoding HSPDE10A, allelicvariants, or related sequences.

Probes may also be used for the detection of related sequences, andshould preferably have at least 50% sequence identity to any of theHSPDE10A encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and may be derived from the sequence of SEQID NO:2 or from genomic sequences including promoters, enhancers, andintrons of the HSPDE10A gene.

Means for producing specific hybridization probes for DNAs encodingHSPDE10A include the cloning of polynucleotide sequences encodingHSPDE10A or HSPDE10A derivatives into vectors for the production of mRNAprobes. Such vectors are known in the art, are commercially available,and may be used to synthesize RNA probes in vitro by means of theaddition of the appropriate RNA polymerases and the appropriate labelednucleotides. Hybridization probes may be labeled by a variety ofreporter groups, for example, by radionuclides such as ³²P or ³⁵S, or byenzymatic labels, such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems, and the like.

Polynucleotide sequences encoding HSPDE10A may be used for the diagnosisof disorders associated with expression of HSPDE10A. Examples of suchdisorders include, but are not limited to, adenocarcinoma, melanoma,myeloma, sarcoma, and teratocarcinoma of the bone, bone marrow, ganglia,gastrointestinal tract, heart, kidney, liver, lung, muscle, nerve,prostate, testis, and uterus and an immune disorder such as adultrespiratory distress syndrome, asthma, atherosclerosis, cholecystitis,Crohn's disease, dermatomyositis, emphysema, erythema nodosum,gastritis, glomerulonephritis, multiple sclerosis, myasthenia gravis,myopathies, osteoarthritis, osteoporosis, polymyositis, rheumatoidarthritis, scleroderma, systemic lupus erythematosus, systemicsclerosis, and ulcerative colitis. The polynucleotide sequences encodingHSPDE10A may be used in Southern or northern analysis, dot blot, orother membrane-based technologies; in PCR technologies; in dipstick,pin, and multiwell formats; and in microarrays utilizing fluids ortissues from patients to detect altered HSPDE10A expression. Suchqualitative or quantitative methods are well known in the art.

In a particular aspect, the nucleotide sequences encoding HSPDE10A maybe useful in assays that detect the presence of associated disorders,particularly those mentioned above. The nucleotide sequences encodingHSPDE10A may be labeled by standard methods and added to a fluid ortissue sample from a patient under conditions for the formation ofhybridization complexes. After an incubation period, the sample iswashed and the signal is quantitated and compared with a standard value.If the amount of signal in the patient sample is significantly alteredin comparison to a control sample then the presence of altered levels ofnucleotide sequences encoding HSPDE10A in the sample indicates thepresence of the associated disorder. Such assays may also be used toevaluate the efficacy of a particular therapeutic treatment regimen inanimal studies, in clinical trials, or to monitor the treatment of anindividual patient.

In order to provide a basis for the diagnosis of a disorder associatedwith expression of HSPDE10A, a normal or standard profile for expressionis established. This may be accomplished by combining body fluids orcell extracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, encoding HSPDE10A, under conditions forhybridization or amplification. Standard hybridization may be quantifiedby comparing the values obtained from normal subjects with values froman experiment in which a known amount of a substantially purifiedpolynucleotide is used. Standard values obtained in this manner may becompared with values obtained from samples from patients who aresymptomatic for a disorder. Deviation from standard values is used toestablish the presence of a disorder.

Once the presence of a disorder is established and a treatment protocolis initiated, hybridization assays may be repeated on a regular basis todetermine if the level of expression in the patient begins toapproximate that which is observed in the normal subject. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

With respect to cancer, the presence of an abnormal amount of transcript(either under- or over-expressed) in biopsied tissue from an individualmay indicate a predisposition for the development of the disease, or mayprovide a means for detecting the disease prior to the appearance ofactual clinical symptoms. A more definitive diagnosis of this type mayallow health professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding HSPDE10A may involve the use of PCR. These oligomersmay be chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably contain a fragment of a polynucleotideencoding HSPDE110A, or a fragment of a polynucleotide complementary tothe polynucleotide encoding HSPDE10A, and will be employed underoptimized conditions for identification of a specific gene or disorder.Oligomers may also be employed under less stringent conditions fordetection or quantitation of closely related DNA or RNA sequences.

Methods which may also be used to quantitate the expression of HSPDE10Ainclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and interpolating results from standard curves(Melby et al. (1993) J Immunol Methods 159:235-244; Duplaa et al. (1993)Anal Biochem 229-236). The speed of quantitation of multiple samples maybe accelerated by running the assay in an multiwell format where theoligomer of interest is presented in various dilutions and aspectrophotometric or colorimetric response gives rapid quantitation.

In further embodiments, oligonucleotides or longer fragments derivedfrom any of the polynucleotide sequences described herein may be used ona microarray. The microarray can be used to monitor the expression levelof large numbers of genes simultaneously and to identify geneticvariants, mutations, and polymorphisms. This information may be used todetermine gene function, to understand the genetic basis of a disorder,to diagnose a disorder, and to develop and monitor the activities oftherapeutic agents.

Microarrays may be prepared, used, and analyzed using methods known inthe art. (See, e.g., Brennan et al. (1995) U.S. Pat. No. 5,474,796;Schena et al. (1996) Proc Natl Acad Sci 93:10614-10619; Baldeschweileret al. (1995) PCT application WO95/251116; Shalon et al. (1995) PCTapplication 095/35505; Heller et al. (1997) Proc Nati Acad Sci94:2150-2155; and Heller et al. (1997) U.S. Pat. No. 5,605,662.)

In another embodiment of the invention, nucleic acid sequences encodingHSPDE10A may be used to generate hybridization probes useful in mappingthe naturally occurring genomic sequence. The sequences may be mapped toa particular chromosome, to a specific region of a chromosome, or toartificial chromosome constructions, e.g., HACs, yeast artificialchromosomes (YACs), bacterial artificial chromosomes (BACs), bacterialP1 constructions, or single chromosome cDNA libraries (Harrington,supra; Price (1993) Blood Rev 7:127-134; and Trask (1991) Trends Genet7:149-154).

Fluorescent in situ hybridization (FISH) may be correlated with otherphysical chromosome mapping techniques and genetic map data(Heinz-Ulrich et al. (1995) In: Meyers, supra, pp.965-968). Examples ofgenetic map data can be found in various scientific journals or at theOnline Mendelian Inheritance in Man (OMIM) site. Correlation between thelocation of the gene encoding HSPDE10A on a physical chromosomal map anda specific disorder, or a predisposition to a specific disorder, mayhelp define the region of DNA associated with that disorder. Thenucleotide sequences of the invention may be used to detect differencesin gene sequences among normal, carrier, and affected individuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques, such as linkage analysis using established chromosomalmarkers, may be used for extending genetic maps. Often the placement ofa gene on the chromosome of another mammalian species, such as mouse,may reveal associated markers even if the number or arm of a particularhuman chromosome is not known. New sequences can be assigned tochromosomal arms by physical mapping. This provides valuable informationto investigators searching for disease genes using positional cloning orother gene discovery techniques. Once the disease or syndrome has beencrudely localized by genetic linkage to a particular genomic region,e.g., ataxia-telangiectasia to 11q22-23, any sequences mapping to thatarea may represent associated or regulatory genes for furtherinvestigation (Gatti et al. (1988) Nature 336:577-580). The nucleotidesequence of the subject invention may also be used to detect differencesin the chromosomal location due to translocation, inversion, deletionand the like among normal, carrier, or affected individuals.

In another embodiment of the invention, HSPDE10A, its catalytic orimmunogenic fragments, or oligopeptides thereof can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes betweenHSPDE10A and the agent being tested may be measured.

Another technique for drug screening provides for high throughputscreening of compounds having binding affinity to the protein ofinterest. (See, e.g., Geysen et al. (1984) PCT application WO84/03564.)In this method, large numbers of different small test compounds aresynthesized on a solid substrate. The test compounds are reacted withHSPDE10A, or fragments thereof, and washed. Bound HSPDE10A is thendetected by methods well known in the art. Purified HSPDE10A can also becoated directly onto plates for use in the aforementioned drug screeningtechniques. Alternatively, non-neutralizing antibodies can be used tocapture the peptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding HSPDE10A specificallycompete with a test compound for binding HSPDE10A. In this manner,antibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with HSPDE10A.

In additional embodiments, the nucleotide sequences which encodeHSPDE10A may be used in any molecular biology techniques that have yetto be developed, provided the new techniques rely on properties ofnucleotide sequences that are currently known, including, but notlimited to, such properties as the triplet genetic code and specificbase pair interactions.

The examples below are provided to illustrate the subject invention andare not included for the purpose of limiting the invention.

EXAMPLES

I cDNA Library Construction

The PROSNOT06 cDNA library was constructed from microscopically normalprostate tissue obtained from a 57-year-old Caucasian male. Pathologyfor the associated tumor indicated adenocarcinoma (Gleason grade 3+3) inboth the left and right periphery of the prostrate. Perineural invasionwas present as was involvement of periprostatic tissue. Patient historyincluded a benign neoplasm of the large bowel, appendectomy, andtonsillectomy with adenoidectomy. Family history included a malignantneoplasm of the prostate and type I diabetes.

The frozen tissue was homogenized and lysed using a POLYTRON homogenizer(PT-3000; Brinkmann Instruments, Westbury, N.J.) in guanidiniumisothiocyanate solution. The lysate was extracted once with acid phenolper Stratagene's RNA isolation protocol (Stratagene, San Diego, Calif.).The RNA was extracted a second time with acid phenol, pH 4.7,precipitated using 0.3 M sodium acetate and 2.5 volumes of ethanol,resuspended in DEPC-treated water, and treated with DNAse at 37° C. for25 minutes. mRNA was isolated with the OLIGOTEX kit (QIAGEN, Chatsworth,Calif.) and used to construct the cDNA libraries. cDNAs werefractionated on a SEPHAROSE CL4B column (APB), and those cDNAs exceeding400 bp were ligated into PSPORT1 plasmid (Life Technologies). Theplasmid was transformed into DH5α competent cells (Life Technologies).

II Isolation of cDNA Clones

Plasmid DNA was released from the cells and purified using the REAL PREP96 plasmid kit (QIAGEN). The recommended protocol was employed exceptfor the following changes: 1) the bacteria were cultured in 1 ml ofsterile TERRIFIC BROTH (BD Biosciences, Sparks Md.) with carbenicillinat 25 mg/l and glycerol at 0.4%; 2) after inoculation, the cultures wereincubated for 19 hours, and the cells were lysed with 0.3 ml of lysisbuffer; and 3) following isopropanol precipitation, the plasmid DNApellet was resuspended in 0.1 ml of distilled water. After the last stepin the protocol, samples were transferred to a 96-well block for storageat 4° C.

III Sequencing and Analysis

The cDNAs were prepared for sequencing using the ABI CATALYST 800thermal cycler (PE Biosystems), or the HYDRA microdispenser (RobbinsScientific) or the MICROLAB 2200 (Hamilton) systems in combination withthe DNA ENGINE thermal cyclers (MJ Research). The cDNAs were sequencedusing the ABI PRISM 373 or 377 sequencing systems and standard ABIprotocols, base calling software, and kits (PE Biosystems) or using theMEGABACE 1000 DNA sequencing system (APB) with solutions and dyes fromAPB. Reading frames were determined using standard methods as reviewedin Ausubel (1997, supra, unit 7.7). Some of the cDNA sequences wereextended using the techniques disclosed in Example V.

The polynucleotide sequences derived from cDNA, extension, and shotgunsequencing were assembled and analyzed using a combination of softwareprograms which utilize algorithms well known to those skilled in theart. Table 2 summarizes the software programs, descriptions, references,and threshold parameters used. The first column of Table 2 shows thetools, programs, and algorithms used, the second column provides a briefdescription thereof, the third column presents the references which areincorporated by reference herein, and the fourth column presents, whereapplicable, the scores, probability values, and other parameters used toevaluate the strength of a match between two sequences (the higher theprobability the greater the homology). Sequences were analyzed usingMACDNASIS PRO software (Hitachi Software Engineering) and LASERGENEsoftware (DNASTAR).

The polynucleotide sequences were validated by removing vector, linker,and polyA sequences and by masking ambiguous bases, using algorithms andprograms based on BLAST, dynamic programming, and dinucleotide nearestneighbor analysis. The sequences were then queried against a selectionof public databases such as GenBank primate, rodent, mammalian,vertebrate, and eukaryote databases, and BLOCKS to acquire annotation,using programs based on BLAST, FASTA, and BLIMPS. The sequences wereassembled into full length polynucleotide sequences using programs basedon Phred, Phrap, and Consed, and were screened for open reading framesusing programs based on GeneMark, BLAST, and FASTA. The full lengthpolynucleotide sequences were translated to derive the correspondingfull length amino acid sequences, and these full length sequences weresubsequently analyzed by querying against databases such as the GenBankdatabases (described above), SwissProt, BLOCKS, PRINTS, PFAM, andProsite.

The programs described above for the assembly and analysis of fulllength polynucleotide and amino acid sequences were used to identifypolynucleotide sequence fragments from SEQ ID NO:2. Fragments from about20 to about 4000 nucleotides which are useful in hybridization andamplification technologies were described in The Invention sectionabove.

IV Cloning of Full Length HSPDE10A

The complete cDNA insert from Incyte clone 826776 was isolated as aSal1/Not1 restriction fragment, labeled with [α-³²P]dCTP, and used as ahybridization probe to screen ˜1×10⁶ plaque forming units from a humanskeletal muscle 5′-STRETCH PLUS λgt10 cDNA library (Clontech, Cat.#HL5002a). Each cDNA insert was recovered as an EcoRI restrictionfragment(s) and subcloned into PBLUESCRIPT KS+ (Stratagene). One λ clone(clone 1a.1) contained a 3.9 kb cDNA insert. Identification of a single,large open reading frame (FIGS. 1A-1E) allowed sequencing of bothstrands to produce the consensus nucleotide sequence, SEQ ID NO:2.HSPDE10A2, a C-terminal splice variant of HSPDE10A2 was also isolated byhybridization screening of the λ Clontech human skeletal muscle cDNAlibrary. When the clone was isolated and fully sequenced, it revealed aninsert with a 5′ coding region similar to HSPDE10A1 and a 3′ end similarto that of the original Incyte clone 826776 (FIGS. 2A-2F).

V Northern Analysis

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7;Ausubel, 1995, supra, ch. 4 and 16.)

Membrane-based northern analysis was performed on RNA samples from avariety of human tissues using Multiple Tissue Northern Blots(Clontech). For detecting human HSPDE10A, the ˜1 kb cDNA insert ofIncyte clone 826776 (Sal1/Not1 restriction fragment) was used. Thiscomprises 108 bp 5′ of the catalytic domain and 429 bp of the catalyticdomain that is common to both HSPDE10A1 and HSPDE10A2. To examineHSPDE10A 1 specifically, the ˜1.7 kb EcoRI restriction fragment of λclone 1a.1 which comprises 447 bp of the 3′ portion of the catalyticdomain and ˜1.2 kb of the 3′ untranslated region was used.

Each probe was labeled with [α-³²P]dCTP using a MEGAPRIME kit (Amersham,Buckinghamshire UK) and reaction products (probe) were purified usingCHROMASPIN-30 columns (Clontech). The Multiple Tissue Northern blotswere pre-hybridized in EXPRESSHYB (Clontech) at 68° C. for 1 hour andhybridized (˜1×10⁶ cpm probe/ml) at 68° C. overnight. Blots were washedin 2×SSPE, 0.05% (w/v) SDS at 50° C. (4×15 mins) followed by 0.1×SSPE,0.1% (w/v) SDS at 50° C. for 1 hour, and then for 2-7 days. Blots werechecked for equal loading of poly(A)⁺ RNA in each lane using a humanβ-actin cDNA probe (data not shown).

Northern analysis showed that HSPDE10A was expressed in skeletal muscleand prostate as a major transcript of ˜7.5 kb; a ˜3.0 kb mRNA wasdetected only in prostate; and a less prominent transcript of ˜1.5 kboccurred in testes and skeletal muscle (FIGS. 5A and 5B). These datasuggest that at least three PDE10A splice variants exist. Electronicnorthern analysis confirms expression in prostate (FIG. 7).

Analogous computer techniques applying BLAST were used to search foridentical or related molecules in nucleotide databases such as GenBankor LIFESEQ database (Incyte Genomics). This analysis is much faster thanmultiple membrane-based hybridizations. In addition, the sensitivity ofthe computer search is modified to determine whether any particularmatch is categorized as exact or similar. The basis of the search is theproduct score, which is defined as:$\frac{\% \quad {sequence}\quad {identity}\quad \times \quad \% \quad {maximum}\quad {BLAST}\quad {score}}{100}$

The product score takes into account both the degree of similaritybetween two sequences and the length of the sequence match. For example,with a product score of 40, the match will be exact within a 1% to 2%error, and, with a product score of 70, the match will be exact. Similarmolecules are usually identified by selecting those which show productscores between 15 and 40, although lower scores identify relatedmolecules.

The results of northern analyses were reported as a percentagedistribution of libraries in which the transcript encoding HSPDE10Aoccurred. Analysis involved the categorization of cDNA libraries byorgan/tissue and disease. The organ/tissue categories includedcardiovascular, dermatologic, developmental, endocrine,gastrointestinal, hematopoietic/immune, musculoskeletal, nervous,reproductive, and urologic. The disease categories included cancer,inflammation/trauma, fetal, neurological, and pooled. For each category,the number of libraries expressing the sequence of interest was countedand divided by the total number of libraries across all categories.Percentage values of tissue-specific and disease expression are reportedin the description of the invention.

VI Labeling and Use of Individual Hybridization Probes

Hybridization probes derived from SEQ ID NO:2 and SEQ ID NO:4 areemployed to screen cDNAs, genomic DNAs, or mRNAs. Although the labelingof oligonucleotides, consisting of about 20 base pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 software (National Biosciences) and labeled bycombining 50 pmol of each oligomer, 250μCi of [³²P]-adenosinetriphosphate (APB), and T4 polynucleotide kinase (NEN Life ScienceProducts, Boston, Mass.). The labeled ligonucleotides are substantiallypurified using a SEPHADEX G-25 superfine size exclusion dextran beadcolumn (APB). An aliquot containing 10⁷ counts per minute of the labeledprobe is used in a typical membrane-based hybridization analysis ofhuman genomic DNA digested with one of the following endonucleases: AseI, Bgl II, Eco RI, Pst I, Xbal, or Pvu II (NEN Life Science Products).

The DNA from each digest is fractionated on a 0.7% agarose gel andtransferred to a NYTRAN PLUS membranes (Schleicher & Schuell, Durham,N.H.). Hybridization is carried out for 16 hours at 40° C. To removenonspecific signals, blots are sequentially washed at room temperatureunder increasingly stringent conditions up to 0.1× saline sodium citrateand 0.5% sodium dodecyl sulfate. After XOMAT-AR film (Eastman Kodak,Rochester, N.Y.) is exposed to the blots to film for several hours,hybridization patterns are compared visually.

VII Microarrays

A chemical coupling procedure and an ink jet device is used tosynthesize array elements on the surface of a substrate (Baldeschweiler,supra). An array analogous to a dot or slot blot is used to arrange andlink elements to the surface of a substrate using thermal, UV, chemical,or mechanical bonding procedures. A typical array is produced by hand orusing available methods and machines and contains any appropriate numberof elements. After hybridization, nonhybridized probes are removed and ascanner used to determine the levels and patterns of fluorescence. Thedegree of complementarity and the relative abundance of each probe whichhybridizes to an element on the microarray is assessed through analysisof the scanned images.

Full-length cDNAs, expressed sequence tags (ESTs), or fragments thereofcomprise the elements of the microarray. Fragments for hybridization areselected using software well known in the art such as LASERGENE software(DNASTAR). Full-length cDNAs, ESTs, or fragments thereof correspondingto one of the nucleotide sequences of the present invention, or selectedat random from a cDNA library relevant to the present invention, arearranged on an appropriate substrate, e.g., a glass slide. The cDNA isfixed to the slide using UV cross-linking followed by thermal andchemical treatments and dried (Schena et al. (1995) Science 270:467-470;Shalon et al. (1996) Genome Res 6:639-645). Fluorescent probes areprepared and used for hybridization to the elements on the substrate.The substrate is analyzed by procedures described above.

VIII Complementary Polynucleotides

Sequences complementary to the HSPDE10A-encoding sequences are used todetect, decrease, or inhibit expression of naturally occurring HSPDE10A.Although use of oligonucleotides comprising from about 15 to 30 basepairs is described, essentially the same procedure is used with smaller(PNA) or with larger fragments. Appropriate oligonucleotides aredesigned using OLIGO 4.06 software (National Biosciences) and the codingsequence of HSPDE10A. To inhibit transcription, a complementaryoligonucleotide is designed from the most unique 5′ sequence and used toprevent promoter binding to the coding sequence. To inhibit translation,a complementary oligonucleotide is designed to prevent ribosomal bindingto the HSPDE10A-encoding transcript.

IX Subcloning and Expression of HSPDE10A

Two constructs encoding full length human HSPDE10Al enzyme (plus andminus an N-terminal epitope tag) were generated for expression in insectcells using a baculovirus vector. Full length human HSPDE10A1 wasisolated by PCR from λ clone 1a.1 using a sense primer,5′-CCAAATCCCGGTCCGAGATGTCCCCAAAGTGCAGTGCTGATGC-3′ (SEQ ID NO:6),covering the initiation codon (underlined) and incorporating an RsrIIrestriction enzyme site, and an antisense primer,5′-CGGGTACCTCGAGTTATTAGTTCCTGTCTTCCTTGGCTACC-3′ (SEQ ID NO:7), coveringthe termination codon (underlined) and incorporating a tandem stop codonand unique XhoI restriction site. PCR was performed using the ExpandHigh Fidelity PCR system (Boehringer Mannheim, West Sussex UK) and thefollowing cycle conditions: 94° C./1′45″, 1 cycle; 94° C./15″, 65°C./30″, 72° C./1′45″, 20 cycles, and 72° C./5′, 1 cycle. The PCR productwas digested with RsrII/XhoI and the resulting restriction fragmentligated into the RsrII/XhoI sites of both the baculovirus transfervector, pFASTBAC (Life Technologies), and with and without modificationto include a 5′ FLAG epitope tag (Kunz et al. (1992) J Biol Chem267:9101-9106). The sequence of the insert for each construct wasdetermined on both strands to confirm identity to the native HSPDE10A1coding sequence, the encoded sequence being either native HSPDE10A1 orN-terminally FLAG-tagged HSPDE10A1.

Recombinant viral stocks were prepared using the Bac-to-Bac system (LifeTechnologies) according to the manufacturer's protocol, and Sf9 cellswere cultured in Sf 900 II serum-free media (Life Technologies) at 27°C. For expression, 3×10⁷ cells in 30 ml were infected at a multiplicityof infection of 1. Cells were harvested 48 hours post-infection forassay. HSPDE10A for enzyme activity assays was prepared from transformedSf9 cells harvested and disrupted by sonication. Cellular debris wasremoved by centrifugation at 12,000×g for 15 mins followed by filtration(0.2 μm filter), and the clarified supernatant dialyzed against 20 mMHEPES pH 7.4, 1 mM EDTA, 150 mM NaCl at 4° C. overnight.

HSPDE10A1 was partially purified from the dialyzed supernatant by ionexchange chromatography using a 1 ml Mono Q HR (5/5) column (APB). Thecolumn was eluted using a linear NaCl gradient up to 1M, and fractionscontaining high activity (>70% substrate turnover) were pooled andstored in aliquots at −70° C.

X PAGE and Western Analysis of HSPDE10A

Transformed Sf9 cells were harvested by centrifugation (3,000×g for 10min), resuspended in homogenization buffer (20 mM HEPES pH 7.2, 1 mMEDTA, 20 mM sucrose, 150 mM NaCl and containing one protease inhibitortablet (Boehringer Mannheim) per 50 ml) at 1×10⁷ cells/ml and disruptedby sonication. Cellular debris was removed by centrifugation at 12,000×gfor 15 min, and the supernatant stored in aliquots at −70° C.

Human PDE10A1 infected and mock infected (control) cell lysates(˜6.4×10⁴ cell equivalents for Coomassie staining, and ˜640 cellequivalents for western analysis) were separated by denaturing PAGEusing the NuPAGE mini-gel system (Novex, San Diego, Calif.) and eitherstained with Coomassie or transferred to a polyvinylidene difluoridemembrane (Novex) for immunoblotting. Western analysis was performed byenhanced chemiluminescence (APB), according to the manufacturer'sprotocol using an anti-FLAG antibody (Sigma-Aldrich, Dorset UK) and ahorse radish peroxidase conjugated anti-mouse IgG (Bio-Rad, Herts UK) asa secondary antibody at 1:500 and 1:1,000 dilutions, respectively.

XI Demonstration of HSPDE10A Activity

PDE activity of HSPDE10A1 was measured using a scintillation proximityassay (SPA)-based method employing the modification reported by Hurwitzet al. (1984; J Biol Chem 259:8612-8618). 50 μl of 20 mM Tris-HCl pH 7.4and 5 mM MgCl₂ containing the required concentration of cyclicnucleotide was added to 50 μl of diluted enzyme (or no enzyme forbackground control) in 20 mM Tris-HCl pH 7.4, 5 mM MgCl₂ and 2 mg/mlbovine serum albumin to initiate the reaction. Both cAMP and cGMP wereused as substrates (0.15-10 μM final concentration) with a 3:1 ratio ofunlabeled to [³ H]-labeled cAMP or cGMP (APB). Reactions were performedin triplicate in MICROFLUOR plates (Dynex Technologies, Chantilly, Va.)at 30° C. for a period of time that would give less than 25% substrateturnover to avoid non-linearity associated with product inhibition. Thereaction was terminated by the addition of 50 μl of PDE SPA beads(yttrium silicate, 20 mg/ml in water; APB) along with a large excess (1mM final concentration) of the respective unlabeled cyclic nucleotide(cGMP or cAMP). Plates were sealed and shaken for 10 minutes to allowthe beads to bind the nucleotide product. The SPA beads were allowed tosettle for 30 minutes, and the plates were read on a TOPCOUNT platereader (Packard Instrument, Meriden, Conn.).

To determine the K_(m) and V_(max) of the enzyme, the rate of hydrolysisof cAMP and cGMP was measured at a variety of substrate concentrations(0.15-10 μM) using a fixed amount of diluted enzyme over a time-courseof 5-60 minutes. Data points in the linear part of the reaction wereused to calculate K_(m) and V_(max) from a Michaelis-Menten plot usingthe ‘Fit Curve’ program of EXCEL software (Microsoft, Redmond Wash.).

Inhibition studies were performed using the assay described above exceptthat the appropriate inhibitor, dissolved and diluted as required indimethylsulphoxide, was added to the diluted enzyme to give the requiredfinal concentration (1-200 μM). Reactions were initiated by the additionof substrate. Cyclic GMP was used as substrate at a final concentrationof 0.17 μM, a concentration equal to ⅓ K_(m) so that IC₅₀˜Ki. Sufficientenzyme was added to give ˜25% substrate turnover during a 30 minuteincubation at 30° C.

XII Functional Assays

HSPDE10A function is assessed by expressing the sequences encodingHSPDE10A at physiologically elevated levels in mammalian cell culturesystems. cDNA is subcloned into pCMV SPORT (Life Technologies) whichcontains the cytomegalovirus promoter. 5-10 μg of recombinant vector 1-2μg of an additional plasmid containing CD64-GFP fusion protein(Clontech) are transiently transformed into a human cell line,preferably of endothelial or hematopoietic origin, using either liposomeformulations or electroporation. Flow cytometry (FCM) is used toidentify transformed cells expressing CD64-GFP. Transformed cells arecollected by contacting the cells with CD64-GFP expressed on theirsurface with magnetic beads coated with either human IgG (DYNAL, LakeSuccess, N.Y.). mRNA is purified from the cells using methods secribedabove and expression of mRNA encoding HSPDE10A is analyzed andquantified by either northern analysis or microarray techniques.

XIII Production of HSPDE10A Specific Antibodies

HSPDE10A purified using PAGE (Harrington (1990) Methods Enzymol182:488-495), is used to immunize rabbits and to produce antibodies.Alternatively, the HSPDE10A amino acid sequence is analyzed usingLASERGENE software (DNASTAR) to determine regions of highimmunogenicity, and an oligopeptide is synthesized and used to raiseantibodies.

Typically, oligopeptides 15 residues in length are synthesized using anABI 431A peptide synthesizer (PE Biosystems) using Fmoc-chemistry andcoupled to KLH (Sigma-Aldrich, St. Louis, Mo.) by reaction withN-maleimidobenzoyl-N-hydroxysuccinimide ester to increase immunogenicity(Ausubel (1995) supra). Rabbits are immunized with the oligopeptide-KLHcomplex in complete Freund's adjuvant. Resulting antisera are tested forantipeptide activity by binding the peptide to plastic, blocking with 1%BSA, reacting with rabbit antisera, washing, and reacting withradio-iodinated goat anti-rabbit IgG.

XIV Purification of Naturally Occurring HSPDE10A Using SpecificAntibodies

Naturally occurring or recombinant HSPDE10A is substantially purified byimmunoaffinity chromatography using antibodies specific for HSPDE10A. Animmunoaffinity column is constructed by covalently couplinganti-HSPDE10A antibody to CNBr-activated SEPHAROSE resin (APB). Afterthe coupling, the resin is blocked and washed according to themanufacturer's instructions.

Media containing HSPDE10A are passed over the immunoaffinity column, andthe column is washed under high ionic strength buffers in the presenceof detergent which allow the preferential absorbance of HSPDE10A. Thecolumn is eluted using a buffer of pH 2 to pH 3 or a high concentrationof a chaotrope, urea or thiocyanate ion, which disruptsantibody/HSPDE10A binding; and HSPDE10A is collected.

XV Identification of Molecules Which Interact with HSPDE10A

HSPDE10A, or biologically active fragments thereof, are labeled with1251 Bolton-Hunter reagent (Bolton et al. (1973) Biochem J 133:529-539).Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled HSPDE10A, washed, and any wellswith labeled HSPDE10A complex are assayed. Data obtained using differentconcentrations of HSPDE10A are used to calculate values for the number,affinity, and association of HSPDE10A with the candidate molecules.

Various modifications and variations of the described methods andsystems of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclaims.

TABLE 1 Selective for PDE type IC50 for HSPDE10A1 Inhibitor (IC50) (μM)IBMX non-selective (2-50 μM) 40 Zaprinast PDE5/6 (0.8/0.2 μM) 8Milrinone PDE3 (1 μM) >200 Rolipram PDE4 (2.0 μM) 160

TABLE 2 Program Description Reference Parameter Threshold ABI A programthat removes vector sequences and Perkin-Elmer Applied Biosystems,FACTURA masks ambiguous bases in nucleic acid sequences. Foster City,CA. ABI/ A Fast Data Finder useful in comparing Perkin-Elmer AppliedBiosystems, Mismatch <50% PARCEL FDF and annotating amino acid ornucleic Foster City, CA; Paracel Inc., Pasadena, CA. acid sequences. ABIAuto- A program that assembles nucleic acid sequences. Perkin-ElmerApplied Biosystems, Assembler Foster City, CA. BLAST A Basic LocalAlignment Search Tool useful in Altschul, S. F. et al. (1990) J. Mol.Biol. ESTs: Probability value = 1.0E-8 sequence similarity search foramino acid and 215:403-410; Altschul, S. F. et al. (1997) or lessnucleic acid sequences. BLAST Nuclec Acids Res. 25:3389-3402. FullLength sequences: Probability includes five functions: blastp, blastn,value = 1.0E-10 or less blastx, tblastn, and tblastx. FASTA A Pearsonand Lipman algorithm that searches for Pearson, W. R. and D. J. Lipman(1988) Proc. ESTs: fasta E value = 1.06E-6 similarity between a querysequence and a group Natl. Acad Sci. 85:2444-2448; Pearson, W. R.Assembled ESTs: fasta Identify = of sequences of the same type. FASTAcomprises (1990) Methods Enzymol. 183:63-98; and 95% or greater andMatch as least five functions: fasta, tfasta, fastx, Smith, T. F. and M.S. Waterman (1981) Adv. length = 200 bases or greater; tfastx, andssearch Appl. Math. 2:482-489. fastx E value = 1.0E-8 or less FullLength sequences: fastx score = 100 or greater BLIMPS A BLocks IMProvedSearcher that matches a Henikoff, S and J. G. Henikoff, Nucl. Acid Score= 1000 or greater; Ratio of sequence against those in BLOCKS and PRINTSRes., 19:6565-72, 1991. J. G. Henikoff and S. Score/Strength = 0.75 orlarger; databases to search for gene families, sequence Henikoff(1996)Methods Enzymol. and Probability value = 1.0E-3 or homology, andstructural fingerprint regions. 266:88-105; and Attwood, T. K. et al.less (1997) J. Chem. Inf. Comput. Sci. 37: 417-424. PFAM A Hidden MarkovModels-based application Krogh, A. et al. (1994) J. Mol. Biol., 235:Score = 10-50 bits, depending on useful for protein family search.1501-1531; Sonnhammer, E. L. L. et al. individual protein families(1988) Nucleic Acids Res. 26:320-322. ProfileScan An algorithm thatsearches for structural Gribskov, M. et al. (1988) CABIOS 4:61-66; Score= 4.0 or greater and sequence motifs in protein sequences Gribskov, etal. (1989) Methods Enzymol. that match sequence patterns defined in183:146-159; Bairoch, A. et al. (1997) Nucleic Prosite. Acids Res. 25:217-221. Phred A base-calling algorithm that examines Ewing, B. et al.(1998) Genome automated sequencer traces with high sensitivity Res.8:175-185; Ewing, B. and P. and probability. Green (1998) Genome Res.8:186- 194. Phrap A Phils Revised Assembly Program including Smith, T.F. and M. S. Waterman (1981) Adv. Score = 120 or greater; Match SWAT andCrossMatch, programs based on Appl. Math. 2:482-489; Smith, T. F. and M.S. length = 56 or greater efficient implementation of the Smith-WatermanWaterman (1981) J. Mol. Biol. 147:195-197; algorithm, useful insearching sequence homology and Green, P., University of Washington, andassembling DNA sequences. Seattle, WA. Consed A graphical tool forviewing and editing Gordon, D. et al. (1998) Genome Phrap assembliesRes. 8:195-202. SPScan A weight matrix analysis program that Nielson, H.et al. (1997) Protein Engineering Score = 5 or greater scans proteinsequences for the presence 10:1-6; Claverie, J. M. and S. Audic (1997)of secretory signal peptides. CABIOS 12:431-439. Motifs A program thatsearches amino acid sequences Bairoch et al. supra; Wisconsin forpatterns that matched those defined in Package Program Manual, versionProsite. 9, page M51-59, Genetics Computer Group, Madison, WI.

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 7 <210> SEQ ID NO 1 <211>LENGTH: 490 <212> TYPE: PRT <213> ORGANISM: HOMO SAPIENS <223> OTHERINFORMATION: HSPDE10A1 <400> SEQUENCE: 1 Met Ser Pro Lys Cys Ser Ala AspAla Glu Asn Ser Phe Lys Glu Ser 1 5 10 15 Met Glu Lys Ser Ser Tyr SerAsp Trp Leu Ile Asn Asn Ser Ile Ala 20 25 30 Glu Leu Val Ala Ser Thr GlyLeu Pro Val Asn Ile Ser Asp Ala Tyr 35 40 45 Gln Asp Pro Arg Phe Asp AlaGlu Ala Asp Gln Ile Ser Gly Phe His 50 55 60 Ile Arg Ser Val Leu Cys ValPro Ile Trp Asn Ser Asn His Gln Ile 65 70 75 80 Ile Gly Val Ala Gln ValLeu Asn Arg Leu Asp Gly Lys Pro Phe Asp 85 90 95 Asp Ala Asp Gln Arg LeuPhe Glu Ala Phe Val Ile Phe Cys Gly Leu 100 105 110 Gly Ile Asn Asn ThrIle Met Tyr Asp Gln Val Lys Lys Ser Trp Ala 115 120 125 Lys Gln Ser ValAla Leu Asp Val Leu Ser Tyr His Ala Thr Cys Ser 130 135 140 Lys Ala GluVal Asp Lys Phe Lys Ala Ala Asn Ile Pro Leu Val Ser 145 150 155 160 GluLeu Ala Ile Asp Asp Ile His Phe Asp Asp Phe Ser Leu Asp Val 165 170 175Asp Ala Met Ile Thr Ala Ala Leu Arg Met Phe Met Glu Leu Gly Met 180 185190 Val Gln Lys Phe Lys Ile Asp Tyr Glu Thr Leu Cys Arg Trp Leu Leu 195200 205 Thr Val Arg Lys Asn Tyr Arg Met Val Leu Tyr His Asn Trp Arg His210 215 220 Ala Phe Asn Val Cys Gln Leu Met Phe Ala Met Leu Thr Thr AlaGly 225 230 235 240 Phe Gln Asp Ile Leu Thr Glu Val Glu Ile Leu Ala ValIle Val Gly 245 250 255 Cys Leu Cys His Asp Leu Asp His Arg Gly Thr AsnAsn Ala Phe Gln 260 265 270 Ala Lys Ser Gly Ser Ala Leu Ala Gln Leu TyrGly Thr Ser Ala Thr 275 280 285 Leu Glu His His His Phe Asn His Ala ValMet Ile Leu Gln Ser Glu 290 295 300 Gly His Asn Ile Phe Ala Asn Leu SerSer Lys Glu Tyr Ser Asp Leu 305 310 315 320 Met Gln Leu Leu Lys Gln SerIle Leu Ala Thr Asp Leu Thr Leu Tyr 325 330 335 Phe Glu Arg Arg Thr GluPhe Phe Glu Leu Val Ser Lys Gly Glu Tyr 340 345 350 Asp Trp Asn Ile LysAsn His Arg Asp Ile Phe Arg Ser Met Leu Met 355 360 365 Thr Ala Cys AspLeu Gly Ala Val Thr Lys Pro Trp Glu Ile Ser Arg 370 375 380 Gln Val AlaGlu Leu Val Thr Ser Glu Phe Phe Glu Gln Gly Asp Arg 385 390 395 400 GluArg Leu Glu Leu Lys Leu Thr Pro Ser Ala Ile Phe Asp Arg Asn 405 410 415Arg Lys Asp Glu Leu Pro Arg Leu Gln Leu Glu Trp Ile Asp Ser Ile 420 425430 Cys Met Pro Leu Tyr Gln Ala Leu Val Lys Val Asn Val Lys Leu Lys 435440 445 Pro Met Leu Asp Ser Val Ala Thr Asn Arg Ser Lys Trp Glu Glu Leu450 455 460 His Gln Lys Arg Leu Leu Ala Ser Thr Ala Ser Ser Ser Ser ProAla 465 470 475 480 Ser Val Met Val Ala Lys Glu Asp Arg Asn 485 490<210> SEQ ID NO 2 <211> LENGTH: 1784 <212> TYPE: DNA <213> ORGANISM:HOMO SAPIENS <223> OTHER INFORMATION: HSPDE10A1 <400> SEQUENCE: 2tggaaagatg ttacttcatc tcccaggttt gctcactgca aatacaatcc tgagaactga 60actagggcct taaagtcctg acatgcatgg cttggttttg tggattgcct ctctcaacag 120gtggtgaaat ttaccaaatc ctttgaattg atgtccccaa agtgcagtgc tgatgctgag 180aacagtttca aagaaagcat ggagaaatca tcatactccg actggctaat aaataacagc 240attgctgagc tggttgcttc aacaggcctt ccagtgaaca tcagtgatgc ctaccaggat 300ccgcgctttg atgcagaggc agaccagata tctggttttc acataagatc tgttctttgt 360gtccctattt ggaatagcaa ccaccaaata attggagtgg ctcaagtgtt aaacagactt 420gatgggaaac cttttgatga tgcagatcaa cgactttttg aggcttttgt catcttttgt 480ggacttggca tcaacaacac aattatgtat gatcaagtga agaagtcctg ggccaagcag 540tctgtggctc ttgatgtgct atcataccat gcaacatgtt caaaagctga agttgacaag 600tttaaggcag ccaacatccc tctggtgtca gaacttgcca tcgatgacat tcattttgat 660gacttttctc tcgacgttga tgccatgatc acagctgctc tccggatgtt catggagctg 720gggatggtac agaaatttaa aattgactat gagacactgt gtaggtggct tttgacagtg 780aggaaaaact atcggatggt tctataccac aactggagac atgccttcaa cgtgtgtcag 840ctgatgttcg cgatgttaac cactgctggg tttcaagaca ttctgaccga ggtggaaatt 900ttagcggtga ttgtgggatg cctgtgtcat gacctcgacc acaggggaac caacaatgcc 960ttccaagcta agagtggctc tgccctggcc caactctatg gaacctctgc taccttggag 1020catcaccatt tcaaccacgc cgtgatgatc cttcaaagtg agggtcacaa tatctttgct 1080aacctgtcct ccaaggaata tagtgacctt atgcagcttt tgaagcagtc aatattggca 1140acagacctca cgctgtactt tgagaggaga actgaattct ttgaacttgt cagtaaagga 1200gaatacgatt ggaacatcaa aaaccatcgt gatatatttc gatcaatgtt aatgacagcc 1260tgtgaccttg gagccgtgac caaaccgtgg gagatctcca gacaggtggc agaacttgta 1320accagtgagt tcttcgaaca aggagatcgg gagagattag agctcaaact cactccttca 1380gcaatttttg atcggaaccg gaaggatgaa ctgcctcggt tgcaactgga gtggattgat 1440agcatctgca tgcctttgta tcaggcactg gtgaaggtca acgtgaaact gaagccgatg 1500ctagattcag tagctacaaa cagaagtaag tgggaagagc tacaccaaaa acgactgctg 1560gcctcaactg cctcatcctc ctcccctgcc agtgttatgg tagccaagga agacaggaac 1620taaacctcca ggtcagctgc agctgcaaaa tgactacagc ctgaagggcc attttcagtc 1680cagcaatgtc atccttttgt tcttttagct cagaaagacc taacatctca aggatgcact 1740gggaaccatg cctgggcttt caccttgaag catggtcagc agca 1784 <210> SEQ ID NO 3<211> LENGTH: 367 <212> TYPE: PRT <213> ORGANISM: HOMO SAPIENS <223>OTHER INFORMATION: HSPDE10A2 <400> SEQUENCE: 3 Met Ser Pro Lys Cys SerAla Asp Ala Glu Asn Ser Phe Lys Glu Ser 1 5 10 15 Met Glu Lys Ser SerTyr Ser Asp Trp Leu Ile Asn Asn Ser Ile Ala 20 25 30 Glu Leu Val Ala SerThr Gly Leu Pro Val Asn Ile Ser Asp Ala Tyr 35 40 45 Gln Asp Pro Arg PheAsp Ala Glu Ala Asp Gln Ile Ser Gly Phe His 50 55 60 Ile Arg Ser Val LeuCys Val Pro Ile Trp Asn Ser Asn His Gln Ile 65 70 75 80 Ile Gly Val AlaGln Val Leu Asn Arg Leu Asp Gly Lys Pro Phe Asp 85 90 95 Asp Ala Asp GlnArg Leu Phe Glu Ala Phe Val Ile Phe Cys Gly Leu 100 105 110 Gly Ile AsnAsn Thr Ile Met Tyr Asp Gln Val Lys Lys Ser Trp Ala 115 120 125 Lys GlnSer Val Ala Leu Asp Val Leu Ser Tyr His Ala Thr Cys Ser 130 135 140 LysAla Glu Val Asp Lys Phe Lys Ala Ala Asn Ile Pro Leu Val Ser 145 150 155160 Glu Leu Ala Ile Asp Asp Ile His Phe Asp Asp Phe Ser Leu Asp Val 165170 175 Asp Ala Met Ile Thr Ala Ala Leu Arg Met Phe Met Glu Leu Gly Met180 185 190 Val Gln Lys Phe Lys Ile Asp Tyr Glu Thr Leu Cys Arg Trp LeuLeu 195 200 205 Thr Val Arg Lys Asn Tyr Arg Met Val Leu Tyr His Asn TrpArg His 210 215 220 Ala Phe Asn Val Cys Gln Leu Met Phe Ala Met Leu ThrThr Ala Gly 225 230 235 240 Phe Gln Asp Ile Leu Thr Glu Val Glu Ile LeuAla Val Ile Val Gly 245 250 255 Cys Leu Cys His Asp Leu Asp His Arg GlyThr Asn Asn Ala Phe Gln 260 265 270 Ala Lys Ser Gly Ser Ala Leu Ala GlnLeu Tyr Gly Thr Ser Ala Thr 275 280 285 Leu Glu His His His Phe Asn HisAla Val Met Ile Leu Gln Ser Glu 290 295 300 Gly His Asn Ile Phe Ala AsnLeu Ser Ser Lys Glu Tyr Ser Asp Leu 305 310 315 320 Met Gln Leu Leu LysGln Ser Ile Leu Ala Thr Asp Leu Thr Leu Tyr 325 330 335 Phe Glu Glu LysVal Arg Asn Thr Ser Pro Gly Ala Val Asn His Leu 340 345 350 Pro Gly ThrSer Asn Leu Gln Leu Phe Phe Gly Ala Pro Pro Tyr 355 360 365 <210> SEQ IDNO 4 <211> LENGTH: 1982 <212> TYPE: DNA <213> ORGANISM: HOMO SAPIENS<223> OTHER INFORMATION: HSPDE10A2 <400> SEQUENCE: 4 tcgacgtggaaagatgttac ttcatctccc aggtttgctc actgcaaata caatcctgag 60 aactgaactagggccttaaa gtcctgacat gcatggcttg gttttgtgga ttgcctctct 120 caacaggtggtgaaatttac caaatccttt gaattgatgt ccccaaagtg cagtgctgat 180 gctgagaacagtttcaaaga aagcatggag aaatcatcat actccgactg gctaataaat 240 aacagcattgctgagctggt tgcttcaaca ggccttccag tgaacatcag tgatgcctac 300 caggatccgcgctttgatgc agaggcagac cagatatctg gttttcacat aagatctgtt 360 ctttgtgtccctatttggaa tagcaaccac caaataattg gagtggctca agtgttaaac 420 agacttgatgggaaaccttt tgatgatgca gatcaacgac tttttgaggc ttttgtcatc 480 ttttgtggacttggcatcaa caacacaatt atgtatgatc aagtgaagaa gtcctgggcc 540 aagcagtctgtggctcttga tgtgctatca taccatgcaa catgttcaaa agctgaagtt 600 gacaagtttaaggcagccaa catccctctg gtgtcagaac ttgccatcga tgacattcat 660 tttgatgacttttctctcga cgttgatgcc atgatcacag ctgctctccg gatgttcatg 720 gagctggggatggtacagaa atttaaaatt gactatgaga cactgtgtag gtggcttttg 780 acagtgaggaaaaactatcg gatggttcta taccacaact ggagacatgc cttcaacgtg 840 tgtcagctgatgttcgcgat gttaaccact gctgggtttc aagacattct gaccgaggtg 900 gaaattttagcggtgattgt gggatgcctg tgtcatgacc tcgaccacag gggaaccaac 960 aatgccttccaagctaagag tggctctgcc ctggcccaac tctatggaac ctctgctacc 1020 ttggagcatcaccatttcaa ccacgccgtg atgatccttc aaagtgaggg tcacaatatc 1080 tttgctaacctgtcctccaa ggaatatagt gaccttatgc agcttttgaa gcagtcaata 1140 ttggcaacagacctcacgct gtactttgag gagaaggtca gaaatacatc acctggagct 1200 gtgaaccacctacctggcac aagcaatctg cagctcttct ttggagcacc cccttattga 1260 tgatggaaagaaccctgtct gtgtctgcct tgatacttgg tattgccttg gtacagcagc 1320 ctgtgatgctgttacatagc atgagggctg ctggccccac tgtccataca cttacaacat 1380 gaaaagctatctggcccaaa ggtttatgct acacatagtt tacaaagatt atctcagagg 1440 gcagaaccgggaggctgggg acttataatc tacccagaag gaaaagttct tccttataga 1500 agatttcaattaacacacat ggaaaggtgg aaatggaaaa atcatcagct ggcaaatacc 1560 acggtagtaatttttattgg caacaataaa tctttctgta actgccctgg gaccttgaac 1620 aagtcacttcaccttccttc accttgagtt tcctcaccta taaaatgaga gaattaatag 1680 gagatttttctcaaaagttc catacagccc taccagtcta taactataat gaaaattcaa 1740 acatagaaaagaagtcattc tatgacccag caattttaca tatacatgta catattcata 1800 tacacagagagagagaactc acacaaattc acaaggaaac atgtacaagg tggttcatag 1860 ctgcattgtatgtaatagca agaaatatta gaaaaatata aattttcatc ttccaggaaa 1920 tgggtaaatagacagtggta taataataga tggaaatagc atacatcagt atgaaggaat 1980 gg 1982<210> SEQ ID NO 5 <211> LENGTH: 875 <212> TYPE: PRT <213> ORGANISM: HOMOSAPIENS <220> FEATURE: <223> OTHER INFORMATION: GI 3355606 <400>SEQUENCE: 5 Met Glu Arg Ala Gly Pro Ser Phe Gly Gln Gln Arg Gln Gln GlnGln 1 5 10 15 Pro Gln Gln Gln Lys Gln Gln Gln Arg Asp Gln Asp Ser ValGlu Ala 20 25 30 Trp Leu Asp Asp His Trp Asp Phe Thr Phe Ser Tyr Phe ValArg Lys 35 40 45 Ala Thr Arg Glu Met Val Asn Ala Trp Phe Ala Glu Arg ValHis Thr 50 55 60 Ile Pro Val Cys Lys Glu Gly Ile Arg Gly His Thr Glu SerCys Ser 65 70 75 80 Cys Pro Leu Gln Gln Ser Pro Arg Ala Asp Asn Ser ValPro Gly Thr 85 90 95 Pro Thr Arg Lys Ile Ser Ala Ser Glu Phe Asp Arg ProLeu Arg Pro 100 105 110 Ile Val Val Lys Asp Ser Glu Gly Thr Val Ser PheLeu Ser Asp Ser 115 120 125 Glu Lys Lys Glu Gln Met Pro Leu Thr Pro ProArg Phe Asp His Asp 130 135 140 Glu Gly Asp Gln Cys Ser Arg Leu Leu GluLeu Val Lys Asp Ile Ser 145 150 155 160 Ser His Leu Asp Val Thr Ala LeuCys His Lys Ile Phe Leu His Ile 165 170 175 His Gly Leu Ile Ser Ala AspArg Tyr Ser Leu Phe Leu Val Cys Glu 180 185 190 Asp Ser Ser Asn Asp LysPhe Leu Ile Ser Arg Leu Phe Asp Val Ala 195 200 205 Glu Gly Ser Thr LeuGlu Glu Val Ser Asn Asn Cys Ile Arg Leu Glu 210 215 220 Trp Asn Lys GlyIle Val Gly His Val Ala Ala Leu Gly Glu Pro Leu 225 230 235 240 Asn IleLys Asp Ala Tyr Glu Asp Pro Arg Phe Asn Ala Glu Val Asp 245 250 255 GlnIle Thr Gly Tyr Lys Thr Gln Ser Ile Leu Cys Met Pro Ile Lys 260 265 270Asn His Arg Glu Glu Val Val Gly Val Ala Gln Ala Ile Asn Lys Lys 275 280285 Ser Gly Asn Gly Gly Thr Phe Thr Glu Lys Asp Glu Lys Asp Phe Ala 290295 300 Ala Tyr Leu Ala Phe Cys Gly Ile Val Leu His Asn Ala Gln Leu Tyr305 310 315 320 Glu Thr Ser Leu Leu Glu Asn Lys Arg Asn Gln Val Leu LeuAsp Leu 325 330 335 Ala Ser Leu Ile Phe Glu Glu Gln Gln Ser Leu Glu ValIle Leu Lys 340 345 350 Lys Ile Ala Ala Thr Ile Ile Ser Phe Met Gln ValGln Lys Cys Thr 355 360 365 Ile Phe Ile Val Asp Glu Asp Cys Ser Asp SerPhe Ser Ser Val Phe 370 375 380 His Met Glu Cys Glu Glu Leu Glu Lys SerSer Asp Thr Leu Thr Arg 385 390 395 400 Glu His Asp Ala Asn Lys Ile AsnTyr Met Tyr Ala Gln Tyr Val Lys 405 410 415 Asn Thr Met Glu Pro Leu AsnIle Pro Asp Val Ser Lys Asp Lys Arg 420 425 430 Phe Pro Trp Thr Thr GluAsn Thr Gly Asn Val Asn Gln Gln Cys Ile 435 440 445 Arg Ser Leu Leu CysThr Pro Ile Lys Asn Gly Lys Lys Asn Lys Val 450 455 460 Ile Gly Val CysGln Leu Val Asn Lys Met Glu Glu Asn Thr Gly Lys 465 470 475 480 Val LysPro Phe Asn Arg Asn Asp Glu Gln Phe Leu Glu Ala Phe Val 485 490 495 IlePhe Cys Gly Leu Gly Ile Gln Asn Thr Gln Met Tyr Glu Ala Val 500 505 510Glu Arg Ala Met Ala Lys Gln Met Val Thr Leu Glu Val Leu Ser Tyr 515 520525 His Ala Ser Ala Ala Glu Glu Glu Thr Arg Glu Leu Gln Ser Leu Ala 530535 540 Ala Ala Val Val Pro Ser Ala Gln Thr Leu Lys Ile Thr Asp Phe Ser545 550 555 560 Phe Ser Asp Phe Glu Leu Ser Asp Leu Glu Thr Ala Leu CysThr Ile 565 570 575 Arg Met Phe Thr Asp Leu Asn Leu Val Gln Asn Phe GlnMet Lys His 580 585 590 Glu Val Leu Cys Arg Trp Ile Leu Ser Val Lys LysAsn Tyr Arg Lys 595 600 605 Asn Val Ala Tyr His Asn Trp Arg His Ala PheAsn Thr Ala Gln Cys 610 615 620 Met Phe Ala Ala Leu Lys Ala Gly Lys IleGln Asn Lys Leu Thr Asp 625 630 635 640 Leu Glu Ile Leu Ala Leu Leu IleAla Ala Leu Ser His Asp Leu Asp 645 650 655 His Arg Gly Val Asn Asn SerTyr Ile Gln Arg Ser Glu His Pro Leu 660 665 670 Ala Gln Leu Tyr Cys HisSer Ile Met Glu His His His Phe Asp Gln 675 680 685 Cys Leu Met Ile LeuAsn Ser Pro Gly Asn Gln Ile Leu Ser Gly Leu 690 695 700 Ser Ile Glu GluTyr Lys Thr Thr Leu Lys Ile Ile Lys Gln Ala Ile 705 710 715 720 Leu AlaThr Asp Leu Ala Leu Tyr Ile Lys Arg Arg Gly Glu Phe Phe 725 730 735 GluLeu Ile Arg Lys Asn Gln Phe Asn Leu Glu Asp Pro His Gln Lys 740 745 750Glu Leu Phe Leu Ala Met Leu Met Thr Ala Cys Asp Leu Ser Ala Ile 755 760765 Thr Lys Pro Trp Pro Ile Gln Gln Arg Ile Ala Glu Leu Val Ala Thr 770775 780 Glu Phe Phe Asp Gln Gly Asp Arg Glu Arg Lys Glu Leu Asn Ile Glu785 790 795 800 Pro Thr Asp Leu Met Asn Arg Glu Lys Lys Asn Lys Ile ProSer Met 805 810 815 Gln Val Gly Phe Ile Asp Ala Ile Cys Leu Gln Leu TyrGlu Ala Leu 820 825 830 Thr His Val Ser Glu Asp Cys Phe Pro Leu Leu AspGly Cys Arg Lys 835 840 845 Asn Arg Gln Lys Trp Gln Ala Leu Ala Glu GlnGln Glu Lys Met Leu 850 855 860 Ile Asn Gly Glu Ser Gly Gln Ala Lys ArgAsn 865 870 875 <210> SEQ ID NO 6 <211> LENGTH: 43 <212> TYPE: DNA <213>ORGANISM: HOMO SAPIENS <400> SEQUENCE: 6 ccaaatcccg gtccgagatgtccccaaagt gcagtgctga tgc 43 <210> SEQ ID NO 7 <211> LENGTH: 41 <212>TYPE: DNA <213> ORGANISM: HOMO SAPIENS <400> SEQUENCE: 7 cgggtacctcgagttattag ttcctgtctt ccttggctac c 41

What is claimed is:
 1. A purified, recombinantly-produced proteincomprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3.
 2. Apurified variant having at least 90% identity to the amino acid sequenceof claim
 1. 3. A composition comprising the protein of claim 1 and apharmaceutical carrier.
 4. A method of using a protein to produce andpurify polyclonal antibodies, the method comprising: a) immunizing ananimal with the protein of claim 1 under conditions to elicit anantibody response; b) isolating animal antibodies; and c) screening theisolated antibodies with the protein to purify antibodies whichspecifically bind the protein.
 5. A method of using a protein to producemonoclonal antibodies, the method comprising: a) immunizing an animalwith the protein of claim 1 under conditions to elicit an antibodyresponse; b) isolating antibody-producing cells from the animal; c)fusing the antibody-producing cells with immortalized cells in cultureto form monoclonal antibody-producing hybridoma cells; d) culturing thehybridoma cells; e) isolating monoclonal antibodies from culture; and f)screening the isolated antibodies with the protein to identifymonoclonal antibodies which specifically bind the protein.
 6. A methodfor using a protein to screen a plurality of molecules or compounds toidentify a ligand, the method comprising: a) combining the protein ofclaim 1 with the molecules or compounds under conditions to allowspecific binding; and b) detecting specific binding, thereby identifyinga ligand which specifically binds the protein.
 7. The method of claim 6wherein the molecules or compounds are selected from DNA molecules, RNAmolecules, peptide nucleic acid molecules, peptides, proteins, agonists,antagonists, antibodies, inhibitors, drug compounds and pharmaceuticalagents.